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Materials
59
Materials
Chemicals and Biochemicals
Proteomic grade fine chemicals were purchased from GE Healthcare
Bio-Sciences, Uppsala, Sweden, USB Cooperation, Ohio, USA, Bio-rad
Laboratories, Hercules, USA and Sigma-Aldrich Co., St. Louis, USA. Immobiline
drystrips and IPG buffers were purchased from GE Healthcare Bio-Sciences,
Uppsala, Sweden. Protein molecular weight marker was procured from GE
Healthcare Bio-Sciences, Uppsala, Sweden. Ampholytes were purchased from
Bio-rad Laboratories, Hercules, USA. Cyanine dyes were purchased from GE
Healthcare Bio-Sciences, Uppsala, Sweden. Peptide-N-Glycosidase F enzyme was
purchased from Sigma-Aldrich Co., St. Louis, USA. Pro-Q®
Diamond
phosphoprotein stain and SYPRO®
Ruby stain were purchased from Molecular
Probes Inc., Oregon, USA. Nitrocellulose membrane was purchased from GE
Healthcare Bio-Sciences, Uppsala, Sweden. Ultrafiltration spin columns were
procured from Pall Life Sciences, Michigan, USA.
Mass spectrometry grade chemicals, solvents and matrices were purchased from
Sigma-Aldrich Co., St. Louis, USA and Merck Specialities Pvt Ltd, Mumbai, India.
Trypsin was purchased from Invitrogen Life Technologies, California, USA. Peptide
mass calibration kit was purchased from Sigma-Aldrich Co., St. Louis, USA.
Solvents and other chemicals of highest purity were procured from Merck
Specialities Pvt Ltd, Mumbai, India and SD Fine Chemicals Pvt Ltd, India. Methanol
was purchased from Sisco Research Laboratories Pvt Ltd, Mumbai, India and
ethanol was distilled out in the laboratory from crude alcohol.
Animals
Inbred strains of immunocompetent Balb/C male and female mice were used as
model organism for M. leprae footpad inoculation.
60
Water quality
Deionized water of resistivity 18.2 m /cm was used for preparation of reagents and
stocks for proteomic experiments. Single and/or double distilled water was used for
routine experiments.
Immunodepletion kits
Qproteome murine albumin depletion kit was purchased from Qiagen, Hilden,
Germany. Chicken IgY spin columns for mouse serum albumin depletion was
purchased from Genway Biotech, San Diego, USA and Chicken IgY spin columns
for removal of Top7 abundant proteins from mouse plasma was purchased from
Sigma-Aldrich Co., St. Louis, USA.
Antibodies
Rabbit anti-haptoglobin antibody was purchased from Dako, Glostrup, Denmark and
HRP labeled goat anti-rabbit IgG antibody was purchased from Genei, Bangalore,
India.
ELISA Kit
Mouse haptoglobin ELISA kit was purchased from Genway Biotech, San Deigo,
USA.
61
SOLUTIONS FOR M. leprae FOOTPAD INOCULATION AND ACID FAST
STAINING
Hank’s Balanced Salt Solution
Potassium chloride - 0.4 g
Disodium hydrogen phosphate - 0.04 g
Potassium dihydrogen phosphate - 0.06 g
Sodium bicarbonate - 0.35 g
Sodium chloride - 8.0 g
Dextrose - 1.0 g
Deionized water to - 900.0 ml
Adjusted the pH to 7.4 and made it upto 1.0 liter with deionized water. Sterilized by
autoclaving and stored as aliquots at 4 ºC.
Carbol Fuschin Stain
Solution A
Basic Fuschin - 0.3 g
95% Ethanol - 10.0 ml
Solution B
Phenol - 5.0 ml
Deionized water - 95.0 ml
Mixed solution A and B and kept at room temperature for several days. The stain
was filtered through Whatman No. 1 filter paper and stored at room temperature in
amber bottles.
Decolorizing agent
Hydrochloric acid - 3.0 ml
Ethanol - 97.0 ml
Acid was added slowly to alcohol and the reagent was stored in amber bottles at
room temperature.
Counter stain
Methylene blue chloride - 0.3 g
62
Deionized water - 100.0 ml
Filtered through Whatman No. 1 filter paper and stored in amber bottles at room
temperature.
SOLUTIONS FOR PROTEIN ESTIMATION
Protein Standard
Stock (10 mg/ml)
Bovine Serum Albumin - 10.0 mg
Deionized water - 1.0 ml
Stored at -20 C as aliquots.
Working Solution (1 mg/ml)
BSA stock - 100.0 l
Deionized water - 900.0 µl
Prepared fresh just before use.
A) Bradford method
Stock
Coomassie brilliant blue G250 - 175.0 mg
Ethanol (95%) - 50.0 ml
Dissolved well and added
Orthophosphoric acid (85%) - 100.0 ml
The stock solution was filtered through Whatman No. 2 filter paper and stored
in amber bottle at room temperature indefinitely.
Working Solution
Bradford stock - 6.0 ml
Orthophosphoric acid (85%) - 6.0 ml
Ethanol (95%) - 3.0 ml
Deionized water - 85.0 ml
Working solution was prepared fresh, just before use, in an amber bottle.
63
B) Lowry’s method
Reagent A
Sodium hydroxide - 2.0 g
Sodium carbonate - 10.0 g
Deionized water - 500.0 ml
Stored at room temperature.
Reagent B
Copper sulfate - 1.0 g
Deionized water - 100.0 ml
Stored at room temperature.
Reagent C
Sodium potassium tartrate - 1.0 g
Deionized water - 100.0 ml
Stored at room temperature.
Alkaline copper reagent
Reagent B - 1.0 ml
Reagent C - 1.0 ml
Reagent A - 98.0 ml
The reagents were mixed in the given order prior to use.
Folin-Ciocalteu solution
Commercially available Folin-Ciocalteu solution (2 N) was diluted 1:1 with
deionized water prior to use.
SOLUTIONS FOR PLASMA PROTEIN DESALTING
A) By gel filtration
Gel filtration media
Sephadex G-25 - 1.0 g
Deionized water - 10.0 ml
Incubated overnight at 4 ºC to swell the matrix before packing the column.
64
Sample diluent
Deionized water was used as sample diluent to dilute plasma samples prior to
gel filtration.
B) By ultra filtration
Nanosep rinsing solution
Ethanol - 700.0 l
Deionized water - 300.0 l
Sample diluent
Deionized water was used as sample diluent to dilute plasma samples prior to
ultra filtration.
C) By precipitation
i) TCA-acetone precipitation
Trichloroacetic acid - 1.0 g
Acetone (chilled) - 10.0 ml
Dissolved TCA in acetone and chilled at -20 ºC for 2-3 h. Just before use,
20 mM DTT was added to the final concentration.
ii) TCA precipitation
Trichloroacetic acid - 11.01 g
Deionized water to - 5.0 ml
Dissolved TCA in deionized water to get 100% saturated TCA solution and
chilled at -20 ºC for 2-3 h prior to use.
iii) Acetone precipitation
Acetone was chilled at -20 ºC over night prior to use.
SOLUTIONS FOR LIQUID PHASE ISOELECTRIC FOCUSING
Anode buffer
Orthophosphoric acid - 636.7 l (0.1 M)
Deionized water to - 100.0 ml
65
Cathode buffer
Sodium hydroxide - 0.4 g (0.1 M)
Deionized water - 100.0 ml
Sample buffer
Urea - 1.26 g
Thiourea - 0.456 g
CHAPS - 0.12 g
1 M DTT - 150.0 l
Ampholyte - 60.0 l
SOLUTIONS FOR MURINE SERUM ALBUMIN DEPLETION
A) By differential extraction
i) Using ethanol
Dilution buffer
1 M Sodium chloride - 400.0 l
1 M HEPES pH 7.4 - 40.0 l
Deionized water - 1.56 ml
Prepared fresh before use.
Chilled ethanol
Ethanol was chilled at -20 ºC for 2-3 h prior to use.
ii) Using 10% TCA-acetone
10% TCA-acetone
Trichloroacetic acid - 1.0 g
Acetone (chilled) - 10.0 ml
Dissolved TCA in acetone and chilled at -20 ºC for 2-3 h. Just before use,
20 mM DTT was added to the final concentration.
B) Using immunoaffinity methods
i) Using rabbit anti-mouse albumin IgG
Dilution/binding buffer for depletion under non-denaturing condition
Sodium dihydrogen phosphate - 0.156 g
66
Sodium chloride - 0.194 g
Deionized water - 20.0 ml
Adjusted the pH to 7.2 and sterilized by autoclaving. Stored at 4 ºC and
brought to room temperature before use.
Dilution/binding buffer for depletion under denaturing condition
Tris - 0.121 g
Urea - 0.24 g
CHAPS - 0.8 g
Deionized water - 20.0 ml
Adjusted the pH to 7.5 and stored at 4 ºC.
ii) Using chicken anti-mouse albumin IgY
Depletion buffer
Tris-HCl - 10 mM
Sodium chloride - 150 mM
pH 7.2
The buffer, supplied as 10X concentrate was diluted to 1X with deionized
water before use. Depletion buffer was supplemented with 80 mM thiourea
or 0.05% CHAPS to reduce the non-covalent interactions in plasma
proteins for some experiments.
Stripping buffer
Glycine - 0.1 M
pH 2.5
The buffer, supplied as 10X concentrate was diluted to 1X with deionized
water before use.
Neutralization buffer
Tris HCl - 0.1 M
pH 8.0
The buffer, supplied as 10X concentrate was diluted to 1X with deionized
water before use.
67
Storage buffer
1X Dilution buffer - 1.0 ml
2% Sodium azide - 10.0 l
SOLUTIONS FOR MURINE ALBUMINOME SEPARATION
Denaturant
10% SDS was added to sample to a final concentration of 0.5%.
10% TCA-acetone
Trichloroacetic acid - 1.0 g
Acetone (chilled) - 10.0 ml
Dissolved TCA in acetone and chilled at -20 ºC for 2-3 h. Just before use, 20 mM
DTT was added to the final concentration.
SOLUTIONS FOR TOP7 MICE PLASMA PROTEIN DEPLETION
Depletion buffer
Tris-HCl - 10 mM
Sodium chloride - 150 mM
pH 7.2
The buffer supplied as 10X concentrate and was diluted to 1X with deionized water
before use.
Stripping buffer
Glycine - 0.1 M
pH 2.5
The buffer supplied as 10X concentrate and was diluted to 1X with deionized water
before use.
Neutralization buffer
Tris HCl - 0.1 M
pH 8.0
The buffer supplied as 10X concentrate and was diluted to 1X with deionized water
before use.
68
Storage buffer
1X Dilution buffer - 1.0 ml
2% Sodium azide - 10.0 l
SOLUTIONS FOR POLYACRYLAMIDE GEL ELECTROPHORESIS
(1D PAGE)
Acrylamide stock (44:0.8)
Acrylamide - 43.2 g
N,N’-Methylenebisacrylamide - 0.8 g
Added 60 ml of deionized water and dissolved well with a magnetic stirrer. Made
upto 100 ml and stored at 4 C in an amber bottle.
Acrylamide stock (30:0.8)
Acrylamide - 29.2 g
N,N’-Methylenebisacrylamide - 0.8 g
Added 60 ml of deionized ware and dissolved well with a magnetic stirrer. Made
upto 100 ml and stored at 4 C in an amber bottle
Separating buffer
Tris base - 18.15 g (1.5 M)
Dissolved in 80 ml of deionized water. Adjusted pH to 8.8 with 2 N HCl. Made upto
100 ml and sterilized by autoclaving.
Stacking buffer
Tris base - 6.05 g (0.5 M)
Dissolved in 80 ml of deionized water. Adjusted pH to 6.8 with 2 N HCl. Made upto
100 ml and sterilized by autoclaving.
10% SDS
Sodium dodecyl sulfate - 10.0 g
Deionized water - 100.0 ml
Stored at room temperature.
10% APS
Ammonium persulfate - 0.1 g
69
Deionized water - 1.0 ml
Prepared fresh prior to use. Diluted to 1% with deionized water for preparation of
separating gel mix.
Separating gel mix (11%)
Acrylamide stock (44:0.8) - 5.0 ml
1.5 M Tris-Cl pH 8.8 - 5.0 ml
10% SDS - 0.4 ml
Deionized water - 9.06 ml
Mixed well. Degassed for 5 min. Chilled on ice for 15 min and then the following
were added.
1% APS - 0.51 ml
TEMED - 0.04 ml
Bottom seal mix
Separating gel mix - 1.0 ml
10 % APS - 10.0 l
TEMED - 5.0 l
Gel overlay solution A
Butanol - 5.0 ml
Deionized water - 5.0 ml
Mixed well and allowed the layers to separate. The upper water saturated butanol
layer was used for overlaying.
Gel overlay solution B
10% SDS - 0.1 ml
1.5 M Tris-Cl pH 8.8 - 2.5 ml
Deionized water to - 10.0 ml
The separating gel was overlaid with overlay solution B solution when left for
overnight aging.
Stacking gel mix (4.5%)
Acrylamide mix (30:0.8) - 0.75 ml
0.5 M Tris-Cl pH 6.8 - 1.25 ml
70
10% SDS - 0.05 ml
Deionized water - 2.92 ml
10% APS - 0.05 ml
TEMED - 0.01 ml
1% Bromophenol blue
Bromophenol blue - 100.0 mg
Tris base - 60.0 mg
Deionized water - 10.0 ml
5X Sample buffer
0.5 M Tris pH 6.8 - 1.72 ml
10% SDS - 2.76 ml
100% Glycerol - 1.38 ml
-mercaptoethanol - 0.69 ml
1% Bromophenol blue - 3.45 ml
Stored as 1 ml aliquots at -20 C.
Running buffer
Tris base - 3.0 g
Glycine - 14.4 g
SDS - 1.0 g
Deionized water to - 1.0 liter
SOLUTIONS FOR TWO DIMENSIONAL GEL ELECTROPHORESIS
(2D PAGE)
UTC concentrate buffer
Urea - 3.6 g (6 M)
Thiourea - 2.3 g (3 M)
CHAPS - 0.8 g (8%)
Added 2-3 ml of deionized water and dissolved completely at 37 C. Made up to
10 ml and stored as 1 ml aliquots at -80 C. The aliquots were brought to room
temperature prior to use and added the following,
1 M DTT - 100.0 µl (100 mM)
71
IPG buffer - 20.0 µl (2%)
UTC rehydration buffer
Urea - 4.2 g (7 M)
Thiourea - 1.52 g (2 M)
CHAPS - 0.4 g (4%)
Added 2-3 ml of deionized water and dissolved completely at 37 C. Made up to
10 ml and stored as 1 ml aliquots at -80 C. The aliquots were brought to room
temperature prior to use and added the following,
1 M DTT - 50.0 µl (50 mM)
IPG buffer - 10.0 µl (1%)
1% BPB - 10.0 µl (0.01%)
Acrylamide mix (44:0.8)
Acrylamide - 55.0 g
N,N’-Methylenebisacrylamide - 1.0 g
Added 80 ml of deionized water and dissolved well with a magnetic stirrer. Made up
to 125 ml.
Acrylamide mix (30:0.8)
Acrylamide - 61.32 g
N,N’-Methylenebisacrylamide - 1.68 g
Added 160 ml of deionized water and dissolved well with a magnetic stirrer. Made
upto 210 ml.
Separating buffer
Tris base - 21.25 g (1.5 M)
Added 100 ml of deionized water. Adjusted pH to 8.8 with 2 N HCl. Made upto
150 ml.
SDS equilibration buffer
1.5 M Tris-Cl pH 8.8 - 4.02 ml
Urea - 43.2 g
Thiourea - 18.0 g
100% Glycerol - 41.2 ml
72
Added 30 ml of deionized water and dissolved completely at 37 C. Made up to
120 ml and then added,
SDS - 2.4 g
Reducing buffer
DTT - 1.2 g (2%)
SDS equilibration buffer - 60.0 ml
Dissolved well by incubating at 37 ºC in water bath and then added,
1% BPB - 50.0 l
Alkylating buffer
IAA - 1.5 g (2.5%)
SDS equilibration buffer - 60.0 ml
Dissolved well by incubating at 37 ºC in water bath and then added,
1% BPB - 50.0 l
12.5% Separating gel
Acrylamide mix (30:08) - 209.0 ml
1.5 M Tris-Cl pH 8.8 - 125.0 ml
Deionized water - 164.0 ml
Mixed well and degassed for 10 min. Chilled on ice for 30 min and then added the
following,
10% APS - 5.0 ml
TEMED - 250 µl
11% Separating gel
Acrylamide mix (44:0.8) - 125.0 ml
1.5 M Tris-Cl pH 8.8 - 125.0 ml
Deionized water - 236.7 ml
Mixed well and degassed for 10 min. Chilled on ice for 30 min and then added the
following,
1% APS - 12.5 ml
TEMED - 800 l
73
9-16% Gradient separating gel
Light solution (9%)
Acrylamide mix (30:0.8) - 75.0 ml
1.5 M Tris-Cl pH 8.8 - 62.5 ml
Deionized water - 111.5 ml
Degassed for 5 min and chilled for 30 min and then added the following
10% APS - 875.0 l
TEMED - 87.5 l
Heavy solution (16%)
Acrylamide mix (30:0.8) - 133.25 ml
1.5 M Tris-Cl pH 8.8 - 62.5 ml
Deionized water - 3.3 ml
Glycerol - 50.0 ml
Degassed for 5 min and chilled for 30 min and then added the following,
10% APS - 875.0 l
TEMED - 87.5 l
Gradient gel preparation for subsequent ammoniacal silver staining
Light solution (9%)
Acrylamide mix (30:0.8) - 75.0 ml
1.5 M Tris-Cl pH 8.8 - 62.5 ml
Deionized water - 110.15 ml
Degassed for 5 min and chilled for 30 min and then added the following
1 M Sodium thiosulfate - 1.25 ml
10% APS - 1.0 ml
TEMED - 100.0 l
Heavy solution (16%)
Acrylamide mix (30:0.8) - 133.25 ml
1.5 M Tris-Cl pH 8.8 - 62.5 ml
Deionized water - 1.9 ml
Glycerol - 50.0 ml
Degassed for 5 min and chilled for 30 min and then added the following,
74
1 M Sodium thiosulfate - 1.25 ml
10% APS - 1.0 ml
TEMED - 100.0 l
10X Running buffer
Tris base - 22.5 g
Glycine - 109.0 g
SDS - 7.5 g
Deionized water to - 750 ml
Diluted 240 ml of 10X stock to 2X concentration for cathode buffer and the
remaining 510 ml was diluted to 1X concentration for anode buffer.
Agarose overlay solution
Agarose - 0.25 g
1X running buffer - 50 ml
Melted by heating on a hot plate and added 50 µl of 1% BPB. Cooled to 50 C
before use.
SOLUTIONS FOR CYANINE DYE LABELING
Cyanine dye solution
Stock
Cyanine dye (Cy2/Cy3/Cy5) - 5 nmol
99.9% Dimethyl formamide - 5.0 µl
Vortexed well and spin down. Final concentration- 1 nmol/µl. Stored at -20 C
upto a month.
Working solution
Cyanine dye stock - 2.0 µl
99.9% Dimethyl formamide - 6.0 µl
Vortexed well and spin down. Final concentration- 125 pmol/µl. Stored at
-20 C upto a week.
1M Tris solution
Tris base - 1.21 g
75
Deionized water - 10.0 ml
Labeling buffer
Urea - 4.2 g (7 M)
Thiourea - 1.52 g (2 M)
CHAPS - 0.4 g (4%)
1 M Tris - 300.0 µl (30 mM)
Added 2-3 ml of deionized water and dissolved completely at 37 C. Adjusted pH to
8.5 with 2 N HCl. Made upto 10 ml and stored as 1 ml aliquots at -80 C. The
aliquots were brought to room temperature prior to use.
Stop solution
Lysine - 9.1 mg
Deionized water - 5.0 ml
Prepared fresh before use.
50 mM Sodium hydroxide
Sodium hydroxide - 20.0 mg
Deionized water - 10.0 ml
SOLUTIONS FOR COLLIODAL COOMASSIE BLUE STAINING
Fixing solution
Methanol - 500.0 ml
Acetic acid - 100.0 ml
Deionized water - 400.0 ml
Colloidal coomassie blue stain
Deionized water - 500.0 ml
85% Orthophosphoric acid - 100.0 ml
Mixed well and then added,
Ammonium sulfate - 100.0 g
Dissolved completely, made up to 800 ml and then added
Coomassie brilliant blue G250 - 1.2 g
Mixed well and then added,
Methanol - 200.0 ml
76
Allowed to age for a few hours before use.
Destaining solution
Destaining was carried out by repeated washes with deionized water.
SOLUTIONS FOR SILVER STAINING
A) Modified Blum method
Fixing solution
Ethanol - 400.0 ml
Acetic acid - 100.0 ml
Deionized water - 500.0 ml
Rinsing solution
Ethanol - 300.0 ml
Deionized water - 700.0 ml
Sensitizing solution
Sodium thiosulfate - 0.2 g
Deionized water - 1.0 liter
Prepared fresh just before use.
Stain
Silver nitrate - 1.0 g
Chilled deionized water - 1.0 liter
Prepared fresh just before use.
Developing solution
Sodium carbonate - 20.0 g
Deionized water to - 1.0 liter
Just before use, added,
37% Formaldehyde - 400.0 µl
Stop solution
Acetic acid - 50.0 ml
Deionized water to - 1.0 liter
77
B) Glutaraldehyde sensitizer method
Fixing solution
Ethanol - 400.0 ml
Acetic acid - 100.0 ml
Deionized water - 500.0 ml
Sensitizing solution
Ethanol - 300.0 ml
Sodium thiosulfate - 2.0 g
Sodium acetate - 68.0 g
Deionized water to - 980.0 ml
Just before use added,
25 % Glutaraldehyde - 20.0 ml
Stain
Silver nitrate - 1.0 g
Deionized water - 1.0 liter
37 % Formaldehyde - 0.4 ml
Formaldehyde was added at the time of use.
Developing solution
Sodium carbonate - 25.0 g
Deionized water to - 1.0 liter
37% Formaldehyde - 0.3 ml
Formaldehyde was added at the time of use.
Stop solution
EDTA - 18.6 g
Deionized water - 1.0 liter
C) Ammoniacal silver method
Fixing solution
Ethanol - 400.0 ml
Acetic acid - 100.0 ml
Deionized water - 500.0 ml
78
Rinsing solution
Ethanol - 50.0 ml
Acetic acid - 50.0 ml
Deionized water - 900.0 ml
Sensitizing solution
Sodium acetate - 41.0 g
25% Glutaraldehyde - 40.0 ml
Deionized water to - 1.0 liter
0.1% Napthalenedisulfonic acid
NDS - 0.5 g
Deionized water - 1.0 liter
Stain
Solution A
Silver nitrate - 8.0 g
Deionized water - 40.0 ml
Solution B
10 N Sodium hydroxide - 2.0 ml
Ammonia - 13.2 ml
Deionized water to - 200.0 ml
Added solution A to B little by little with constant stirring to dissolve the
brown precipitate formed. Made up to 1000 ml with deionized water.
Developing solution
10% Citric acid - 2.0 ml
Deionized water to - 1.0 liter
At the time of use added,
37% Formaldehyde - 2.7 ml
Stop solution
Tris base - 50.0 g
Acetic acid - 20.0 ml
Deionized water to - 1.0 liter
79
SOLUTIONS FOR PHOSPHOPROTEIN STAINING
Fixer
Methanol - 50.0 ml
Acetic acid - 10.0 ml
Deionized water - 40.0 ml
Stain
Pro-Q®
Diamond phosphoprotein stain was used as supplied.
Destaining solution
1 M Sodium acetate pH 4.0 - 5.0 ml
Acetonitrile - 20.0 ml
Deionized water - 75.0 ml
SOLUTIONS FOR SYPRO® RUBY STAINING
Fixer
Methanol - 100.0 ml
Acetic acid - 20.0 ml
Deionized water - 80.0 ml
Stain
SYPRO®
Ruby stain was diluted 1:1 with deionized water and used for staining.
Destaining solution
Methanol - 10.0 ml
Acetic acid - 7.0 ml
Deionized water - 83.0 ml
SOLUTIONS FOR MADLI-TOF MASS SPECTROMETRY
A) Destaining solution
25 mM Ammonium bicarbonate in 50% ACN
Ammonium bicarbonate - 20.0 mg
100% Acetonitrile - 5.0 ml
Deionized water - 5.0 ml
80
Prepared fresh just before use.
B) Solutions for tryptic digestion
100 mM Ammonium bicarbonate in 10% ACN
Ammonium bicarbonate - 8.0 mg
Acetonitrile - 100.0 l
Deionized water - 900.0 l
Prepared fresh just before use.
Trypsin
Stock (1 g/ l)
Trypsin - 25.0 g
50 mM Acetic acid - 25.0 l
Stored as 5 l aliquots at -80 ºC.
Working solution (80 ng/ l)
Trypsin stock - 5.0 l
100 mM Ammonium bicarbonate
in 10% ACN - 57.5 l
Prepared fresh just before use.
Overlay solution
Ammonium bicarbonate - 3.2 mg (40 mM)
Acetonitrile - 100.0 l (10%)
Deionized water - 900.0 l
Prepared fresh just before use.
C) Extraction solution
5% Trifluoroacetic acid - 20.0 l (0.1%)
100% Acetonitrile - 600.0 l (60%)
Deionized water - 380.0 l
Prepared fresh just before use.
D) Resuspension solution
Acetonitrile - 5.0 l (5%)
5% Trifluoroacetic acid - 2.0 l (0.1%)
81
Deionized water - 93.0 l
Prepared fresh just before use.
E) Solutions for zip-tip purification
Wetting and elution solution
Acetonitrile - 500.0 l (50%)
5% Trifluoroacetic acid - 20.0 l (0.1%)
Deionized water - 480.0 l
Prepared fresh and aliquoted 50 l each for wetting and 5 l each for elution.
Equilibration and washing solution
5% Trifluoroacetic acid - 20.0 l (0.1%)
Deionized water - 980.0 l
Prepared fresh and aliquoted 50 l each for equilibration and washing.
F) Matrix
CHCA (10 mg/ml)
-Cyano 4-hydroxy cinnamic acid - 10 mg
100 % Acetonitrile - 500.0 l (50%)
5% Trifluoroacetic acid - 20.0 l (0.1%)
Deionized water - 480.0 l
Dissolved the matrix in the solvent thoroughly by vortexing. Stored at 4 ºC
as 100 l aliquots. The vials were covered with aluminium foil to protect
from light.
DHB (50 mg/ml)
Dihydroxy benzoic acid - 50.0 mg
Acetone - 990.0 l (99%)
Deionized water - 10.0 l
Dissolved the matrix in solvent by vortexing. Prepared fresh just before use.
G) Solutions for peptide mix preparation
Bradykinin stock (100 pmol/ l)
Bradykinin fragment 1-7 - 10 nmol
50% ACN in 0.05% TFA - 100.0 l
82
Stored at -20 ºC. Diluted to 10 pm/ l working concentration with 50% ACN
in 0.05% TFA.
Angiotensin stock (100 pmol/ l)
Angiotensin II - 10 nmol
0.1% TFA - 100.0 l
Stored at -20 ºC. Diluted to 10 pm/ l working concentration with 0.1% TFA.
P14R stock (100 pmol/ l)
P14R - 10 nmol
0.1% TFA - 100.0 l
Stored at -20 ºC. Diluted to 10 pm/ l working concentration with 0.1% TFA.
ACTH stock (100 pmol/ l)
ACTH (18-39) - 10 nmol
0.1% TFA - 100.0 l
Stored at -20 ºC. Diluted to 10 pm/ l working concentration with 0.1% TFA.
Peptide mix
10 pmol/ l Bradykinin - 9.0 l
10 pmol/ l Angiotensin - 4.0 l
10 pmol/ l P14R - 3.0 l
10 pmol/ l ACTH - 3.0 l
100% ACN:0.1% TFA (30:70) - 1.0 l
Stored at -20 ºC.
SOLUTIONS FOR LC-MS/MS ANALYSIS
A) Peptide resuspension solution
Formic acid - 1.0 l (0.1%)
Deionized water - 999.0 l
B) Chromatography solution A
Formic acid - 500.0 l (0.1%)
HPLC grade water - 499.5 ml
83
C) Chromatography solution B
Acetonitrile - 400.0 ml (80%)
Formic acid - 400.0 l (0.8%)
HPLC grade water - 99.6 ml
SOLUTIONS FOR WESTERN BLOTTING
Towbin buffer
Tris base - 0.54 g
Glycine - 0.27 g
SDS - 0.035 g
Deionized water to - 80.0 ml
The solution was chilled at 4 ºC for 2 h and just before use added,
Methanol - 20.0 ml
1 M Tris pH 7.5
Tris base - 12.1 g
Deionized water - 75.0 ml
Adjusted the pH to 7.5 with 2 N HCl and made upto 100 ml. The solution was
sterilized by autoclaving and stored at 4ºC.
Tris buffered saline (TBS)
1 M Tris-Cl pH 7.5 - 5.0 ml
Sodium chloride - 4.5 g
Deionized water to - 500.0 ml
Tris buffered saline-Tween (TBS-T)
1 M Tris-Cl pH 7.5 - 5.0 ml
Sodium chloride - 4.5 g
Deionized water to - 500.0 ml
Tween-20 - 0.5 ml
Blocking buffer
Skim milk powder - 1.0 g (5%)
TBS-T - 20.0 ml
84
Primary antibody (1:1000 dilution)
TBS - 20.0 ml
Skim milk powder - 20.0 mg
Primary antibody - 20.0 l
Secondary antibody (1:2000 dilution)
TBS - 20.0 ml
Skim milk powder - 20.0 mg
Secondary antibody - 10.0 l
50 mM Tris pH 7.5
1 M Tris-Cl pH 7.5 - 2.5 ml
Deionized water to - 50.0 ml
Developing solution
Detection reagent A
Nickel chloride - 15.0 mg (0.03%)
50 mM Tris-Cl pH 7.5 - 5.0 ml
Detection reagent B
3, 3’ Diaminobenzidine - 30.0 mg (0.06%)
50 mM Tris-Cl pH 7.5 - 45.0 ml
The reagent was prepared fresh and care was taken to protect from light. Just
before use mixed reagent A and B and added
30% Hydrogen peroxide - 50.0 l
The developing solution was used immediately.
SOLUTIONS FOR ELISA
Stock solutions provided in mouse haptoglobin ELISA kit were diluted to the
required concentration and used for assay. The reagents were stored at 4 ºC
and were brought to room temperature before assay.
Mouse haptoglobin standard (14.25 g/ml)
Lyophilized mouse haptoglobin
calibrator - 1 vial
85
Deionized water - 1.0 ml
Stored at -20 ºC as 100 l aliquots.
Sample diluent
5X concentrate - 10.0 ml
Deionized water - 40.0 ml
Wash solution
20X concentrate - 25.0 ml
Deionized water - 475.0 ml
Enzyme-antibody conjugate
100X concentrate - 50.0 l
1X sample diluent to - 5.0 ml
Chromogen-substrate solution
A mixture of 3, 3’, 5, 5’ –tetramethylbenzidine (TMB) and hydrogen peroxide
in citrate buffer at pH 3.3 was provided as chromogen-substrate solution and it
was used as supplied.
Stop solution
0.3 M Sulfuric acid was provided as stop solution and it was used as supplied.
SOLUTIONS FOR DEGLYCOSYLATION ASSAY
Denaturation buffer
10% SDS - 40.0 l
1 M -mercaptoethanol - 200.0 l
Deionized water - 1760.0 l
Prepared fresh just before use.
Reaction buffer
Ammonium bicarbonate - 3.6 mg
Deionized water - 2.0 ml
Prepared fresh just before use.
PNGaseF
86
Lyophilized powder of PNGaseF in 5 mM potassium phosphate buffer was
resupsended in deionized water to get 0.5 U/ l enzyme solution. The enzyme was
stored at -20 ºC as aliquots.
87
List of Instruments and Softwares Used in This Study
Table 1: List of instruments
Instrument Model/Manufacturer
2D PAGE unit
Ettan™ DALTsix electrophoresis unit,
GE Healthcare, Sweden; Mini-Protean® 3
Dodeca™ Cell, Bio-rad, USA
Analytical and top loading balances
Model 2474 and L310, Sartorius,
Germany; M220, Denver instruments
Company, USA
Autoclave 20 h/BE, Nat Steel Equipments Pvt. Ltd.,
India
Centrifuges
Megafuge 1.0R, Heraeus, Kendro
Laboratory Products, Germany; 05PR-22,
Hitachi Koki Co., Ltd., Japan
Circulating water bath Model TEWPA, Scigenics Biotech Pvt.
Ltd., India
Concentrator Speedvac concentrator, Savant
Instruments Inc., USA
Cooling water bath MultiTemp III, Amersham Biosciences,
USA
Electroblotting apparatus Semi-phor™, Hoefer Scientific
Instruments, San Francisco
ELISA plate washer Immunowash Model 1575, Bio-rad, USA
ELISA reader Model 680, Bio-rad, USA
End-over-end shaker Rocking Platform Mixer, Ratek
Instruments, Australia
Freezer (-20 ºC) Tropicalised, Blue star Ltd., India
Hot air oven Biochem, Universal Biochemicals, India
Hot plates Hotop, Tarsons Products Pvt Ltd., India
Ice machine SLF 190 A-Q, Blue star Ltd., India
Isoelectric focusing unit Ettan™ IPGphor™, GE Healthcare,
Sweden
Laminar air flow Cleanair, Atlantis, India
88
Liquid phase isoelectric focusing unit MicroRotofor™ Cell, Bio-rad, USA
Magnetic stirrer Ratek Instruments, Australia
MALDI target plate dryer Kratos Analytical Unit, Manchester, UK
Mass spectrometer (MALDI-TOF MS) Kratos Axima CFR plus, Schimadzu,
Japan
Mass spectrometer (Nano LC-MS/MS)
MicrOTOF-Q mass spectrometer, Bruker
Daltonics, Germany, coupled to Ultimate
3000, Dionex, Hong Kong, liquid
chromatography system
Microcentrifuges
Sigma 1-15K and Sigma® 2M, Sigma-Svi
Biosolutions Pvt. Ltd., Germany; Biofuge
pico, Heraeus, Kendro Laboratory
Products, Germany
Multichannel laser scanner Typhoon 9400 Variable Mode Imager,
GE Healthcare, Sweden
One dimensional SDS PAGE units
SE 400 Sturdier, SE 600 and Hoefer
miniVe Vertical slab gel electrophoresis
units, Amersham Biosciences, USA;
SE 450 Mighty Small II, Hoefer Scientific
Instruments, San Francisco
Orbital shaker Greiner Bio-One, UK
pH meter Control Dynamics, Bangalore, India
Power packs
EPS-601, Amersham Biosciences, USA;
PowerPac™ HV, Bio-Rad, USA; Model
4000, BRL Life Technologies Inc., USA;
Biotech Electrophoretic power supply,
Biotech R & D Laboratories, India
Reciprocal shaker Recipro-shake, Luckham, England;
Orbitek, Scigenics (India) Pvt. Ltd., India
Scanner Scanjet G3010, Helwett-Packard
Development Company, India
Sonicator Vibra Cell™, Sonics and Materials Inc.,
USA
89
Ultra low freezer (-80 ºC)
Revco, Legaci™ Refrigeration System,
Copeland® Dupont Suva® Refrigerants,
USA
UV-Vis spectrophotometer U-2000, Hitachi Ltd. Japan
Vacuum pump Gel Pump GP100, Savant Instruments
Inc., USA
Vortex mixer Spinix, Tarsons Products Pvt Ltd., India;
Ratek Instruments, Australia
Water circulated cooling unit Multitemp III, Amersham Biosciences,
USA
Water deionization unit Milli-Q®, Millipore, India
Water distillation unit Labo rota 20, Heidolph, Germany; Model
10S/DE, Nat Steel Equipment Pvt. Ltd.
Water bath sonicator Sonica® Ultrasonic cleanser, Model
2200MH, Soltec, Italy
Table 2: List of softwares
Software Version/Manufacturer
2DE image analysis ImageMaster Platinum 2D Version 7.0,
GE Healthcare, Sweden
2D DIGE image analysis DeCyder™ 2D 7.0, GE Healthcare,
Sweden
2D DIGE extended data analysis DeCyder™ EDA 7.0, GE Healthcare,
Sweden
Densitometry ImageQuant TL, Amersham Biosciences,
USA
90
Methods
91
Methods
1. MOUSE FOOTPAD INOCULATION OF Mycobacterium leprae
M. leprae purified from biopsy samples of leprosy patients were used as the source
of inoculum for footpad injections. Punch biopsies were collected from the leprosy
patients attending the outpatient department in Central Leprosy Teaching and
Research Institute, Chengalpet, Tamil Nadu, India. Sample collection was carried
out as per the guidelines of Institutional Ethical Committee. Approximately
1-2 grams of tissue was collected from the lesions after informed consent. The tissue
was minced well under aseptic conditions and resuspended in 2 ml of Hank’s
balanced salt solution. Further homogenization of the tissue was carried out in
Mickle tissue disintegrator by subjecting to vibration. The bacilli were separated
from the tissues by centrifugation. The supernatant containing M. leprae was
collected and the number of acid fast bacilli per gram of the biopsy tissue was
counted.
Approximately 5 l of the suspension was placed in the centre of each circle in
the ring slide and spread evenly using a wire loop. The smear was heat fixed by
placing the slide on a heater for a few seconds. The smear was flooded with carbol
fuschin for 5 min and washed with water extensively to remove excess stain. The
smear was then decolorized with acid alcohol for 30 sec. The decolorized smear was
washed with water and counterstained with methylene blue for 1 min. The slides
were washed with water to remove excess stain and dried completely. The acid fast
bacilli were counted under 100X oil immersion objective using a compound
microscope. Fifteen fields were counted per circle in the ring slide.
M. leprae footpad inoculation was carried out as described earlier [Levy and Ji,
2006]. In brief, 5 x 103
acid fast bacilli suspended in 30 µl Hank’s balanced salt
solution were inoculated into both the hind footpads using a tuberculin syringe. A
group of 20 mice were inoculated with M. leprae purified from a single biopsy
material and this group was denoted as a batch. The list of samples used for this
study is provided in table M.1. The footpad inoculated mice were maintained under
controlled conditions of temperature, light and humidity in Animal House Facility at
92
Table M.1: List of samples used for plasma proteome analysis
A) Experimental samples
No Batch
ID
No of
AFB per
gram of
biopsy
Treatment
regimen of
patient
Mouse
strain
Sex and
age of the
mouse
No of AFB
injected
per
footpads
1 110 1.39x105 Dapsone Balb/C /3 months 5x103
2 111 1.12x106 Dapsone Balb/C /3 months 5x103
3 112 1.62x106 MDT Balb/C /3 months 5x103
4 113 1.86x106 MDT Balb/C /3 months 5x103
5 114 8.57x106 MDT Balb/C /3 months 5x103
6 115 6.52x106 MDT Balb/C /3 months 5x103
7 116 2.58x106 Dapsone Balb/C /3 months 5x103
8 117 2.47x106 MDT Balb/C /3 months 5x103
9 118 5.31x106 MDT Balb/C /3 months 5x103
10 119 4.85x106 Untreated Balb/C /3 months 5x103
11 120 3.8x104 Untreated Balb/C /3 months 5x103
12 121 2.03x105 MDT Balb/C /3 months 5x103
13 122 4.5x106 Dapsone Balb/C /3 months 5x103
14 123 3.62x106 Untreated Balb/C /3 months 5x103
15 124 5.07x104 MDT Balb/C /3 months 5x103
16 125 7.6x104 MDT Balb/C /3 months 5x103
17 126 9.01x105 MDT Balb/C /3 months 5x103
18 127 1.56x106 Untreated Balb/C /3 months 5x103
19 128 8.34x106 Untreated Balb/C /3 months 5x103
20 129 1.67x107 Untreated Balb/C /3 months 5x103
21 130 3.82x106 Untreated Balb/C /3 months 5x103
22 131 4.58x106 Untreated Balb/C /3 months 5x103
23 132 1.75x106 Dapsone Balb/C /3 months 5x103
24 133 3.86x106 Untreated Balb/C /3 months 5x103
25 134 1.66x106 Dapsone Balb/C /3 months 5x103
93
Table M.1: Continued.
B) Control samples
No Batch ID Mouse
strain
Sex and age of
the mouse
1 B/C 1 Balb/C /9-10 months
2 B/C 2 Balb/C /9-10 months
3 B/C 3 Balb/C /9-10 months
4 B/C 4 Balb/C /9-10 months
5 B/C 5 Balb/C /9-10 months
6 B/C 6 Balb/C /9-10 months
7 B/C 7 Balb/C /9-10 months
8 B/C 8 Balb/C /9-10 months
9 B/C 9 Balb/C /9-10 months
10 B/C 10 Balb/C /9-10 months
11 B/C 11 Balb/C /9-10 months
12 B/C 12 Balb/C /9-10 months
13 B/C 13 Balb/C /9-10 months
14 B/C 14 Balb/C /9-10 months
15 B/C 15 Balb/C /9-10 months
16 B/C 1 Balb/C /9-10 months
17 B/C 2 Balb/C /9-10 months
18 B/C 3 Balb/C /9-10 months
19 B/C 4 Balb/C /9-10 months
20 B/C 5 Balb/C /9-10 months
21 B/C 6 Balb/C /9-10 months
22 B/C 7 Balb/C /9-10 months
23 B/C 8 Balb/C /9-10 months
24 B/C 9 Balb/C /9-10 months
25 B/C 10 Balb/C /9-10 months
94
CLTRI, Chengalpet. Commercially available formulated mice feed was provided to
the animals, throughout the study. Footpad harvesting was carried out after 6, 9 and
12 months of inoculation. Two mice from each batch were sacrificed and the hind
footpads were harvested. The bacilli, purified from each footpad were resupsended
in Hank’s balanced salt solution and the number of acid fast bacilli per footpad was
determined. The bacillary count of 105 was considered as significant multiplication.
All animal experimentations were conducted as per the guidelines of Institutional
Animal Ethical Committee.
2. PLASMA COLLECTION
Blood samples were collected from mouse by retro orbital vein puncture into tubes
containing 10% EDTA as anti-coagulant. Blood samples were collected at 6, 9 and
12 months post footpad inoculation. Plasma was separated by centrifugation at
2000 rpm for 10 min at 4 ºC in a conical bottom tube. Plasma samples were snap
frozen and transported in liquid nitrogen. The plasma samples were frozen in liquid
nitrogen for long term storage whereas aliquots were stored in -80 ºC for immediate
use.
3. TOTAL PROTEIN ASSAY
3.1 Bradford method
Total protein assay by Bradford method [Bradford, 1976] was carried out as follows.
The protein sample was made upto 1 ml with deionized water in clean glass tubes.
Five milliliters of Bradford working solution was added to each tube and mixed well.
The tubes were incubated for 5 min at room temperature in dark. Absorbance was
read at 595 nm after blanking to zero.
3.2 Lowry’s method
Total protein assay by Lowry’s method [Lowry et al., 1951] was carried out as
follows. The protein sample was made upto 1 ml with deionized water in clean glass
tubes. Four milliliters of alkaline copper reagent was added to each tube and mixed
well. The tubes were incubated at room temperature for 10 min. After the incubation,
0.5 ml of Folin’s Ciocalteu reagent was added to the tubes and mixed well. The tubes
95
were incubated in dark for 20 min. Absorbance was read at 750 nm after blanking to
zero.
4. PLASMA DESALTING METHODS
4.1 By gel filtration
Gel filtration was carried out in sephadex G-25 packed spin columns. The empty
spin column was washed in deionized water twice. The hydrated sephadex G-25
matrix was transferred to the column using a blunt end pipette tip. The packed
column was washed twice with 500 l each of deionized water. It was centrifuged at
300 x g for 2 min at 4 ºC to drain the column dry. The plasma sample (10 l) was
diluted to 500 l with deionized water and loaded onto the column. After mixing
well by inverting, the column was centrifuged at 300 x g for 2 min at 4 ºC to collect
the deionized plasma sample as flow through. The protein content in the flow
through fraction was estimated and the samples were analyzed by 1D PAGE and
compared with neat plasma. Average percentage recovery was calculated as per the
formula,
4.2 By ultrafiltration
Ultrafiltration was carried out using nanosep centrifugal filters with 3 kDa molecular
weight cut-off. Nanosep columns were first filled with rinsing solution and incubated
at room temperature for 2 h. The columns were centrifuged at 13800 x g, 25 ºC till
dead stop. Further, the columns were washed with deionized water twice. Ten
microliters of plasma sample was diluted to 600 l with deionized water and added
to nanosep column. The column was subjected to centrifugation at 13800 x g, 25 ºC
till volume was reduced to 50 l. Retentate and filtrate were collected separately and
the protein content was estimated. The samples were analyzed by 1D PAGE and
compared with neat plasma. Average percentage recovery was calculated as
mentioned before.
Concentration of protein recovered
Concentration of protein loaded X 100
96
4.3 By protein precipitation methods
4.3.1 TCA-acetone precipitation
Plasma samples were diluted 50 fold with deionized water before precipitation. The
samples were mixed with two volumes of chilled 10% TCA-acetone containing
20 mM DTT in a 2 ml microcentrifuge tube and incubated at -20 ºC for 1 h. The
precipitated proteins were collected by centrifuging at 12000 rpm for 15 min at 4 ºC.
The supernatant was discarded and the pellet was washed twice with one volume of
chilled acetone to remove the residual TCA. The pellet was air dried for 2-3 min and
resuspended in UTC rehydration buffer. The protein content was estimated and the
samples were analyzed by 1D PAGE and 2D PAGE. Average percentage recovery
was calculated as mentioned before.
4.3.2 TCA precipitation
Plasma samples were diluted 50 fold with deionized water before precipitation. To
the samples, TCA was added to a final concentration of 13% and mixed well. The
samples were incubated at -20 ºC for 5 min followed by incubation on ice for
35 min. The precipitated proteins were collected by centrifugation at 12000 rpm for
20 min at 4 ºC. The supernatant was discarded and the pellet was air dried for
2-3min. The precipitated proteins were resuspended in UTC rehydration buffer. The
protein content was estimated and the samples were analyzed by 1D PAGE. Average
percentage recovery was calculated as mentioned before.
4.3.3 Acetone precipitation
Plasma samples were diluted 50 fold with deionized water before precipitation. The
samples were mixed with two volumes of chilled acetone in a 2 ml microcentrifuge
tube. The tubes were incubated on ice for 15 min and centrifuged at 12000 rpm for
10 min at 4 ºC to recover the precipitated proteins. The supernatant was discarded
and the pellet was air dried for 2-3 min. The pellet was resuspended in UTC
rehydration buffer. The protein content was estimated and the samples were
analyzed by 1D PAGE and 2D PAGE. Average percentage recovery was calculated
as mentioned before.
97
5. ALBUMIN DEPLETION BY DIFFERENTIAL EXTRACTION
5.1 By ethanol treatment
Removal of albumin by ethanol treatment was carried out as described by Fu et al.,
[2005]. To 100 l of mice plasma sample, 100 l of dilution buffer was added and
incubated at 4 ºC for 1 h in an orbital shaker. To this, chilled ethanol was added to a
final concentration of 42%, mixed well and incubated at 4 ºC for 1 h in an orbital
shaker. The precipitated proteins were recovered by centrifugation at 16000 x g for
45 min at 4 ºC. The pellet was air dried for 2-3 min and resuspended in 100 l of
UTC rehydration buffer. The proteins in the supernatant were precipitated using
chilled acetone as described in section 4.3.3. The pellet and supernatant fractions
were analyzed by 1D PAGE and 2D PAGE.
5.2 By 10% TCA-acetone treatment
The plasma sample was diluted 10 fold with deionized water and denatured by
treating with 0.5% SDS at room temperature for 30 min. To this, two volumes of
chilled 10% TCA-acetone containing 20 mM DTT was added and mixed well,
followed by incubation at -20 ºC for 1 h. The precipitated proteins were recovered by
centrifugation at 12000 rpm for 15 min at 4 ºC. The supernatant was saved
separately and the pellet was washed twice with one volume of chilled acetone. The
pellet was air dried and resuspended in UTC rehydration buffer. The proteins present
in the supernatant were recovered by precipitating with chilled acetone as described
in section 4.3.3. The pellet and supernatant fractions were analyzed by 1D PAGE
and 2D PAGE.
6. ALBUMIN DEPLETION BY IMMUNOAFFINITY METHODS
6.1 Using rabbit anti-mouse albumin antibody
Immunoaffinity depletion of mouse serum albumin using rabbit anti-mouse albumin
antibody was carried out using Qproteome murine albumin depletion spin columns
(Qiagen) as per manufacturer’s instructions. Twenty five microliters of mice plasma
(~1 mg) was diluted to 100 l with dilution buffer. The storage buffer in the spin
column was drained by gravity flow and the column was washed twice with 500 l
98
of dilution buffer. The spin column was sealed with a luer plug and the diluted
plasma sample was added. The column was subjected to vigorous shaking to mix the
plasma sample with the matrix and incubated on an end-over-end shaker for 5 min at
room temperature. The luer plug was removed and the spin column was transferred
to a 2 ml collection tube and centrifuged at 500 x g for 10 sec to collect the unbound
plasma proteins in flow through fraction. The column was washed twice with 100 l
each of dilution buffer and the wash fractions were pooled with the flow through. If
depletion was carried out using non-denaturing buffers, the depleted sample was
taken for acetone precipitation as described in section 4.3.3 to remove salts
introduced by the dilution buffer.
6.2 Using chicken anti-mouse albumin antibody
Immunoaffinity depletion of mice serum albumin using chicken anti-mouse albumin
antibody was carried out using Seppro®
IgY mouse albumin depletion spin column
as per manufacturer’s instructions.
Preparation of plasma sample
Approximately 2 mg of mice plasma (50 l) was diluted to 500 l using 1X depletion
buffer. The samples were mixed well by vortexing and centrifuged at 5000 rpm for
10 min at 4 ºC. The supernatant was collected and used for depletion.
Pretreatment of column
The tip of the IgY spin column was snapped off and the column was placed in a 2 ml
collection tube. The column was centrifuged at 2000 rpm for 30 sec at 4 ºC to drain
the storage buffer and washed thrice with 500 l each of depletion buffer to remove
unbound IgY antibodies, if any.
Immunocapture of albumin
Diluted plasma sample was added to the column and mixed thoroughly by shaking
and inverting. The column was incubated in an orbital shaker for 15 min at room
temperature. The end cap was removed and the column was placed in a fresh 2 ml
collection tube. Centrifugation was carried out at 2000 rpm for 30 sec at 4 ºC to
99
collect the unbound proteins as the flow through. The column was washed with
500 l of depletion buffer for maximum recovery of unbound proteins.
Elution of bound albumin
Bound proteins were eluted from the column immediately after immunodepletion.
The column was washed thrice with 500 l each of 1X depletion buffer to remove
the proteins bound to the microbeads by nonspecific interaction. The wash fractions
were collected by centrifuging at 2000 rpm for 30 sec at 4 ºC. The bound albumin
was stripped in four steps by treating the microbeads with stripping buffer. For each
step, 500 l of stripping buffer was added to the column, mixed by shaking and
inverting and incubated in an orbital shaker for 3 min. The bound proteins were
eluted by centrifuging at 2000 rpm for 30 sec at 4 ºC. Eluates from two consecutive
steps were pooled and neutralized with 100 l of neutralizing buffer.
Neutralization of column
The microbeads were neutralized immediately after elution of the bound proteins to
regenerate the column for subsequent rounds of albumin depletion. The beads were
resuspended in 600 l of neutralization buffer and incubated for 5 min in an orbital
shaker. The column was centrifuged at 2000 rpm for 30 sec at 4 ºC to drain the
buffer. The beads were resuspended in storage buffer and the column was stored at
4 ºC until further use.
Reusing the column for immunodepletion
The column was reused for depletion of albumin from mice plasma following the
method described above. The microbeads were transferred to a fresh spin column
after 20 depletion reactions to avoid clogging of the membranes in spin column.
Analysis of fractions
The protein content in prewash fractions, flow through, wash fractions, eluate
fractions and neutralization wash fraction was estimated and these fractions were
analyzed by 1D PAGE. The flow through fraction was subjected to acetone
precipitation as described in section 4.3.3 and resuspended in UTC rehydration
buffer. This step helped to concentrate and to remove the salts introduced by
100
depletion buffer prior to 2DE. The recovery percentage of albumin depleted plasma
was calculated as per the formula,
7. PREPARATION OF MURINE ALBUMINOME
Immunoaffinity capture of albumin from mice plasma was achieved onto anti-mouse
albumin IgY as described in section 6.2. Along with albumin, other proteins
interacting with albumin were also retained in the bound fraction in this method. The
bound proteins were eluted from the column by treating with 0.1 M glycine pH 2.5
and used to purify murine albuminome. The first and second eluate were pooled and
neutralized with neutralization buffer as described in section 6.2. The samples were
then denatured by treating with 0.5% SDS for 30 min at room temperature. The
denatured samples were precipitated by treating with 10% TCA-acetone as described
in section 5.2.
8. DEPLETION OF TOP7 HIGH ABUNDANT PLASMA PROTEINS BY
IMMUNOAFFINITY METHODS
Top7 high abundant proteins in mice plasma viz., albumin, IgG, IgM, fibrinogen,
transferrin, alpha-1-antitrypsin and haptoglobin, were removed in one-step method
using Seppro®
IgY top7 depletion column as per manufacturer’s instructions.
Approximately 500 g of mice plasma proteins (10 l) was diluted to 500 l with
depletion buffer and centrifuged at 5000 rpm for 10 min at 4 ºC to collect the
supernatant. The storage buffer in the spin column was drained by centrifuging at
2000 rpm for 30 sec at 4 ºC. The column was washed thrice with 500 l each of
depletion buffer. The diluted plasma sample was added to the column and mixed
well by inverting and shaking the column. The column was incubated in an orbital
shaker for 30 min at room temperature. The unbound proteins were collected by
centrifugation at 2000 rpm for 30 sec at 4 ºC. The column was washed with 500 l of
depletion buffer for maximum recovery of unbound proteins. The nonspecifically
bound proteins were removed from the microbeads by washing thrice with 500 l
each of depletion buffer. The bound proteins were stripped in 8 steps by treating
Concentration of protein in flow through
Concentration of proteins taken for depletion X 100
101
with stripping buffer immediately after immunodepletion. For each step, 500 l of
stripping buffer was added to the column and incubated in an orbital shaker for
3 min at room temperature. The bound proteins were collected by centrifugation at
2000 rpm for 30 sec at 4 ºC and the eluate fractions from consecutive steps were
pooled and neutralized with 100 l each of neutralization buffer. The microbeads
were neutralized by incubating in 600 l of neutralization buffer for 5 min in an
orbital shaker. The neutralization buffer was drained by centrifugation at 2000 rpm
for 30 sec at 4 ºC and the beads were resuspended in 600 l of storage buffer. The
column was stored at 4 ºC until further use. The column was reused for subsequent
depletions as described before and the microbeads were transferred to a fresh spin
column after every 20 depletion reactions. The flow through fraction was
precipitated with chilled acetone as described in section 4.3.3 and resuspended in
UTC rehydration buffer prior to 2DE.
9. LIQUID PHASE ISOELECTRIC FOCUSING
Equilibration of ion exchange membranes
Cation exchange membrane (anode membrane) was equilibrated by incubating it in
25 ml of 0.1 M orthophosphoric acid at room temperature overnight. Anion
exchange membrane (cathode membrane) was equilibrated by incubating it in 25 ml
of 0.1 M sodium hydroxide at room temperature overnight.
Assembling the focusing unit
Equilibrated anode and cathode membranes were rinsed in distilled water and fitted
onto either sides of the focusing chamber. The anode and cathode buffer chambers
were fixed onto the focusing chamber and aligned the loading ports in focusing
chamber and electrolyte chamber in straight line. The ports at opposite end of the
loading port (harvesting port) were sealed with a tape.
Sample preparation and loading
Plasma sample was diluted 1:1 with UTC concentrate buffer containing 100 mM
DTT and 2% ampholytes. The solution was made upto 3 ml with UTC rehydration
buffer containing 50 mM DTT and 1% ampholytes. The ampholytes were chosen
102
based on the required pH range for separation. Using a syringe, the sample was
loaded carefully through the loading port avoiding air bubbles. The excess solution
was wiped off and the loading ports were sealed with a tape.
Loading the assembly
The focusing assembly was fixed onto the cooling block in Microrotofor with the
loading ports of electrolyte chamber facing up. Free movement of the assembly was
checked before loading the electrolytes. Using syringe, 0.1 M orthophosphoric acid
was added to the anode chamber and 0.1 M sodium hydroxide to the cathode
chamber. The lid was closed and the unit was connected to power supply.
Electrofocusing
Focusing was carried out at 1 W at 20 ºC until the current stabilizes (takes
approximately 2 h).
Harvesting focused fractions
The focusing assembly was removed from the cooling block after focusing. The tape
from loading port was removed and the focusing assembly was placed on harvesting
block. Microrotofor was connected to a vacuum pump and the harvesting tray was
kept in the collecting position. Focusing assembly was pressed onto the pins on the
harvesting block while applying vacuum. Ten fractions (~250 l each) were
collected in tray. The collection tray was removed from the unit after turning the
vacuum off. The fractions were subjected to acetone precipitation and analyzed on a
1D PAGE.
10. SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (1D PAGE)
Laemmli method [1970] was followed for separation of proteins by 1D PAGE as
described earlier [Ausubel et al., 1989]. Proteins were resolved on 0.75 mm or 1 mm
thick resolving gel using vertical slab gel electrophoresis units. The glass plates were
treated with 10% nitric acid and rinsed with distilled water before casting gel. The
cassette was assembled and the bottom seal mix was poured through the sides and
allowed to polymerize. The required volume of separating gel mix was poured into
the sealed cassette and overlaid with gel overlay solution A. The gel was allowed to
103
polymerize for 2 h at room temperature. The overlay solution was removed and
rinsed with deionized water. The gel surface was overlaid with gel overlay solution
B and left for overnight aging at 4 ºC. The gels were brought to room temperature
before casting the stacking gel. The overlay solution was removed and rinsed the gel
surface with deionized water. Appropriate thickness comb was inserted and stacking
solution was poured through the sides, avoiding air bubbles. The stacking gel was
allowed to polymerize for 30 min at room temperature. After that, the comb was
removed and wells were washed with 1X running buffer. The protein samples were
boiled in sample buffer and loaded onto the wells. Anode and cathode chambers
were filled with 1X running buffer. Electrophoresis was carried out at 10 mA
constant current for mini gels (Hoefer miniVe and SE 450 Mighty Small II) and at
15 mA for larger gels (SE 450 and SE 600).
11. TWO DIMENSIONAL GEL ELECTROPHORESIS (2D PAGE)
Two dimensional gel electrophoresis was carried out as described elsewhere [Gupta
et al., 2007].
Preparation of samples
Samples were precipitated with chilled acetone as mentioned in section 4.3.3 before
two dimensional separation. The precipitate was resuspended in UTC rehydration
buffer and the protein content was estimated by Bradford method. The protein
loading concentration for IEF depends on the pH range and length of the IPG strip
and also on the staining method to be employed (Table M.2). The sample was
diluted to the required volume using UTC rehydration buffer containing 50 mM
DTT, 1% IPG buffer and 0.01% BPB.
Rehydration
Immobiline dry strips were rehydrated with rehydration solution. The maximum
volume of buffer that can be used for rehydration was decided based on length of
IPG strip (Table M.3).
Three types of rehydration methods were employed in this study.
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Table M.2: Suitable protein loading concentration for IEF
Length pH range Protein load ( g)
Silver staining CBB staining
7 cm 3-10 NL 3-6 25-60
4-7 L 4-8 25-150
11cm 3-10 NL 7-15 50-120
4-7 L 10-20 50-300
13 cm 3-10 NL 10-20 50-240
4-7 L 15-30 75-450
18 cm 3-10 NL 20-40 100-500
4-7 L 30-60 150-900
Table M.3: Rehydration volume for isoelectric focusing
Length Volume
7 cm 125 l
11 cm 200 l
13 cm 250 l
18 cm 320 l
i) Active rehydration
In this method, the samples in rehydration buffer were pipetted into the strip holder
and the IPG strip was placed over it (gel side facing down) without trapping air
bubbles. The strips were overlaid with covering fluid and rehydration was carried out
at low voltage (30 V) for 11 h before focusing.
ii) Passive rehydration
The samples in rehydration buffer were pipetted into the slots in reswelling tray. The
IPG strips were placed over it without trapping air bubbles. The slots were filled
with 3.5 ml of covering fluid and rehydration was carried out for 16 h at room
temperature. The rehydrated strips were transferred to IPG strip holder for focusing.
105
iii) Cup loading
IPG strips were rehydrated without the protein sample in rehydration buffer in
reswelling tray for 16 h at room temperature. The IPG manifold was placed on the
IPGPhor unit and 8 ml of covering fluid was added in each slot. The rehydrated
strips were placed in the slots, gel side facing up. The position of the strip in the slot
was adjusted to ensure the acidic end of the strip comes in contact with the cathode
and the basic end of the strip comes in contact with the anode. Paper wicks, soaked
in deionized water, were placed on either ends of the strip. The electrodes were
placed over the wicks and locked the cams to complete the circuit. The cups were
placed towards anodic/cathodic end of the strip. The sample in rehydration buffer
(100 l) was pipetted into sample cups for focusing.
Electrofocusing
The rehydrated strips were subjected to electrofocusing at 20 ºC. The strips were
exposed to high voltage using a predefined program as given below. The programs
were chosen based on the pH range and length of the strip and the rehydration
method followed. The maximum current that can be applied per strip was restricted
to 50 A.
Active rehydration; 3-10 NL, 7 cm IPG strips
0 V Rehydration 1 h
Step 1 30 V Step-n-Hold 11 h
Step 2 300 V Step-n-Hold 2 h
Step 3 1000 V Gradient 1 h
Step 4 5000 V Gradient 1.5 h
Step 5 5000 V Step-n-Hold 4 h
Active rehydration; 4-7 L, 11cm IPG strips
0 V Rehydration 1 h
Step 1 30 V Step-n-Hold 11 h
Step 2 200 V Step-n-Hold 2 h
Step 3 1000 V Gradient 1 h
Step 4 6000 V Gradient 2 h
Step 5 6000 V Step-n-Hold 4 h
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Active rehydration; 3-10 NL, 18 cm IPG strips
0 V Rehydration 1 h
Step 1 30 V Step-n-Hold 11 h
Step 2 300 V Step-n-Hold 2 h
Step 3 1000 V Gradient 1 h
Step 4 8000 V Gradient 1.5 h
Step 5 8000 V Step-n-Hold 9 h
Active rehydration; 4-7 L, 18 cm IPG strips
0 V Rehydration 1 h
Step 1 30 V Step-n-Hold 11 h
Step 2 300 V Step-n-Hold 2 h
Step 3 1000 V Gradient 1 h
Step 4 8000 V Gradient 3 h
Step 5 8000 V Step-n-Hold 8 h
Passive rehydration; 3-10 NL, 7 cm IPG strips
Step 1 300 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 5000 V Gradient 1 h
Step 4 5000 V Step-n-Hold 4 h
Passive rehydration; 3-10 NL, 18 cm IPG strips
Step 1 500 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 8000 V Gradient 3 h
Step 4 8000 V Step-n-Hold 9 h
Cup loading; 3-10 NL, 13 cm IPG strips
Step 1 300 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 5000 V Gradient 2 h
Step 4 5000 V Step-n-Hold 6 h
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Cup loading; 4-7 L, 13 cm IPG strips
Step 1 300 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 5000 V Gradient 2 h
Step 4 5000 V Step-n-Hold 6 h
Cup loading; 3-10 NL, 18 cm IPG strips
Step 1 500 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 8000 V Gradient 3 h
Step 4 8000 V Step-n-Hold 5 h
Cup loading; 4-7 L, 18 cm IPG strips
Step 1 500 V Step-n-Hold 1 h
Step 2 1000 V Gradient 1 h
Step 3 5000 V Gradient 3 h
Step 4 5000 V Step-n-Hold 5 h
The strips were either taken for second dimension immediately after focusing or
stored at -80 ºC till the next step.
Equilibration of focused proteins
The strips were brought to room temperature for 30 min. The strips were equilibrated
with reducing buffer for 15 min on an end-over-end shaker followed by incubation in
alkylating buffer for 15 min.
Second dimension PAGE
Six gels were run together for second dimensional separation using Ettan Daltsix
electrophoresis apparatus. Single percentage gels or gradient gels were used for the
second dimension.
Casting method for single percentage gels
Six gel cassettes were assembled and arranged in multiple gel caster, with each
cassette separated by a mylar sheet. The required volume of separating gel (12.5% or
11%) was taken in a reservoir chamber and connected to top filling port in multiple
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gel caster through a silicone tubing. The flow rate was set at 50 ml/min. The gel
cassettes were allowed to fill leaving about 1 cm gap from the top and then overlaid
with gel overlay solution B. The gels were left for polymerization at room
temperature for 2 h and aged overnight at 4 ºC.
Casting method for gradient gels
Gradient gels (9-16%) were used to improve the separation of high molecular weight
proteins. The exact volume required to fill the cassettes arranged in multiple gel
caster was measured by filling with water. The volume was divided equally for light
solution (9%) and heavy solution (16%) and the solutions were chilled for 30 min on
ice. The gel cassettes were assembled as mentioned before and the caster was
connected to gradient maker through a silicone tube. Light solution was taken in
mixing chamber of the gradient maker and the heavy solution in reservoir chamber.
Casting was carried out at a flow rate of 25 ml/min. The gel surface was overlaid
with gel overlay solution A and allowed to polymerize for 2 h at room temperature.
The overlay solution was removed after polymerization and the gel surface was
washed with deionized water prior to electrophoresis. If the gels were to be used for
ammoniacal silver staining, 5 mM sodium thiosulfate was incorporated in the gel
mix. Since thiosulfate retards polymerization, the concentration APS and TEMED
were increased in the gel mix.
Electrophoresis
The gels were brought to room temperature and the cassettes were washed with
deionized water. The overlay solution was removed with a syringe and the gel
surface was washed with deionized water. The equilibrated strip was layered on top
of the gel carefully avoiding air bubbles. The gaps were sealed with agarose overlay
solution. The cassettes were arranged in the Ettan Daltsix electrophoresis apparatus.
The lower buffer tank (anode) was filled with 5 liters of 1X running buffer whereas
the upper buffer tank (cathode) was filled with 1.2 liters of 2X running buffer.
Electrophoresis was carried out at 1 W/gel for 1 h and later at 13 W/gel till dye front
reached the bottom of the separating gel.
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12. TWO DIMENSIONAL DIFFERENCE GEL ELECTROPHORESIS
(2D DIGE)
Two dimensional difference gel electrophoresis was carried out as described below.
Preparation of samples for labeling
Plasma samples were precipitated with chilled acetone as described in section 4.3.3.
The precipitate was resuspended in DIGE labeling buffer. The pH of the samples
was checked and adjusted to 8.5 with 1-2 l of 50 mM sodium hydroxide, wherever
necessary. Protein content was estimated by Bradford method and the concentration
was adjusted to 3 g/ l with labeling buffer. The resolution of proteins was assessed
by 1D PAGE prior to labeling.
Reconstitution of cyanine dyes
Cyanine dyes were reconstituted as per manufacturer’s instructions. In brief, 5 nmol
of the lyophilized powder was brought to room temperature and 5 l of 99.9% DMF
was added to each vial. Mixed well by vortexing vigorously and spun down. Two
microliters of this dye stock was diluted to 8 l with DMF to make the working
solution of concentration 125 pmol/ l.
Preparation of pooled internal standard
Equal concentration of proteins from each of the biological replicates of control and
experimental samples were pooled to generate an internal standard [Alban et al.,
2003]. Thirty micrograms each of the pooled internal standard was aliquoted into
low binding tubes for labeling with Cy2. A flowchart representing the preparation of
pooled internal standard and 2D DIGE work flow is provided in figure M.1.
Labeling
The protein-dye ratio for all the labeling reactions was kept constant at 250 pmol of
dye for 30 g of protein. Thirty micrograms of the protein samples from each
biological replicate of control and experimental samples were taken in duplicates. To
one set of the samples, 2 l of Cy3 was added while to the other 2 l of Cy5 was
added. Two microliters of Cy2 was added to each of the pooled internal standard
110
Image analysis Mass spectrometry
Internal standard
Control Experimental
Cy5 labelingCy3 labeling
Cy2 labeling
Pooling
Two dimensional separation
Imaging
Cy3 Cy5
Cy2
Quantification of differential
expression Protein identification
Fig M.1: A schematic diagram representing the 2D DIGE workflow
111
aliquots. The tubes were vortexed vigorously, spun down and incubated on ice for
30 min. The tubes were covered with aluminium foil to protect from light. The
labeling reaction was stopped by adding 1 l of 10 mM lysine solution to each tube
followed by vigorous vortexing. The tubes were incubated on ice for 10 min. The
labeled samples were diluted with equal volume of UTC concentrate buffer.
Isoelectric focusing
The labeled samples to be separated together were pooled before isoelectric
focusing. For example, an aliquot internal standard (Cy2 labeled), an aliquot of
control sample (Cy3 labeled) and an aliquot of experimental sample (Cy5 labeled)
were pooled. The volume was made upto 100 l with UTC rehydration buffer. The
samples were applied onto a pre-rehydrated strip employing anodic cup loading
method as described in section 11. The electrofocusing was carried out as per the
focusing programs mentioned in section 11.
Second dimension PAGE
The focused strips were reduced with DTT and alkylated with IAA as mentioned
earlier. Second dimensional separation was carried out using 11% polyacrylamide
gel as described in section 11. The gels were protected from light during the second
dimension PAGE. The dye front was allowed to run out of the gel to remove the
unbound cyanine dyes.
Scanning
The gels were separated from the cassette and rinsed with deionized water to remove
SDS. The multichannel laser scanner was given a warm up time of 30 min before
scanning. The location of the gel was specified in the scanner control and the image
was acquired in fluorescent mode. The excitation and emission wave length used for
each cyanine dye is provided in table M.4. Different photomultiplier tube voltages
(520-600V) were used for scanning to avoid supersaturation. The images were stored
as .gel files in 100 microns pixel size. These files were exported to DeCyder™ 2D
software for image analysis.
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Table M.4: Scanning parameters for cyanine dyes
Cy dye ex (nm) em (nm) Laser Band pass PMT (V)
Cy 2 488 520 Blue 40 540
Cy 3 534 580 Red 30 520
Cy 5 633 670 Green 30 540
Counter staining
The gels were counter stained with colloidal coomassie blue or silver nitrate as given
in section 13.
13. STAINING METHODS
13.1 Colloidal coomassie blue staining
Colloidal Coomassie blue staining was carried out as described earlier [Candiano et
al., 2004]. The gels were incubated in fixer solution for 1-2 h and washed thrice with
deionized water for 10 min each to remove excess of acetic acid. The gels were
incubated in Colloidal coomassie blue stain overnight on a shaker and destained by
repeated washing with deionized water.
13.2 Silver staining
13.2.1 Modified Blum method
The gels were stained with silver nitrate employing modified Blum method if the
proteins were to be taken for identification by mass spectrometry [Mortz et al.,
2001]. The gels were fixed for 1 h and incubated in rinsing solution for 20 min. The
gels were sensitized with 0.02% sodium thiosulfate for 1 min and washed thrice with
deionized water for 20 sec each to remove excess sensitizer. Chilled silver nitrate
stain was added onto the gels and incubated at 4 ºC for 30 min on a shaker. The
staining solution was discarded and the gels were rinsed with deionized water for
10 sec. The gels were developed with developer solution till the spots appear. The
developer was replaced with fresh solution every time it turned yellow. Developing
reaction was stopped with 5% acetic acid solution to prevent background staining.
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The gels were washed with water thoroughly (3 x 10 min) to remove the traces of
stop solution to avoid bleaching of spots.
13.2.2 Glutaraldehyde sensitizer method
The gels were subjected to silver staining using glutaraldehyde as sensitizer
[Heukeshoven and Dernick, 1985], when the downstream analysis did not include
mass spectrometry. The gels were fixed for 1 h and sensitized for 30 min in
sensitizing solution containing 0.125% glutaraldehyde. The gels were washed thrice
in deionized water for 15 min each and stained with silver nitrate for 30 min at room
temperature. The stain was poured off and the gels were washed twice with
deionized water to remove the unbound silver nitrate. The gels were developed with
developer solution till the spots appear. The developer was replaced with fresh
solution every time it turned yellow. Developing reaction was stopped with 0.05 M
EDTA solution to prevent background staining. The gels were washed thrice with
water for 10 min each to remove the traces of stop solution to avoid bleaching of
spots.
13.2.3 Ammoniacal silver staining method
Ammoniacal silver staining method [Rabilloud, 1999] is a highly sensitive silver
staining method routinely used to detect proteins from 2D gel when the loading
concentration was extremely low (< 20 g). In order to improve the sensitivity and
reduce the background staining, 5 mM sodium thiosulfate was incorporated into the
acrylamide mix if the gels are to be taken for ammoniacal silver staining. The gels
were fixed for 1 h and then rinsed for 2 h with rinsing solution. The gels were
washed with water for 5 min and incubated in sensitizer for 30 min. Excess
glutaraldehyde in the sensitizer was removed by washing thrice with deionized water
for 10 min each. The gels were further incubated in 0.1% NDS for 30 min and
washed with deionized water for 4 times, 15 min each. The gels were then stained
with ammoniacal silver nitrate solution for 30 min. The stain was discarded and the
gels were washed with deionized water 4 times for 4 min each. The gels were
developed with developer solution for 1-2 min or till the spots appear. Developing
was stopped with stop solution to prevent background staining. The gels were rinsed
in water thoroughly to remove the traces of stop solution to avoid bleaching of spots.
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13.3 SYPRO® Ruby staining
SYPRO®
Ruby staining [Berggren et al., 2000] was carried out as per
manufacturer’s instructions. The gels were transferred to plastic trays and treated
with fixer solution for 15 min. The fixer was replaced with fresh fixing solution and
incubated for another 15 min to achieve maximum removal of SDS from the gels.
The gels were incubated in stain overnight in dark. After staining, the gels were
transferred to a fresh tray and destained with destaining solution twice for 30 min
each followed by washing with deionized water for 1 h. The image was acquired by
exciting the fluorophores at 488 nm and the emission was recorded at 610 nm. The
digitized images were saved in ‘.gel’ format.
14. PHOSPHOPROTEIN STAINING OF MICE PLASMA PROTEINS
Phosphorylation in mice plasma proteins was analyzed by staining with Pro-Q®
Diamond, a fluorescent phosphoprotein stain specific for serine, threonine and
tyrosine phosphorylation. The gels were taken in a glass tray and incubated in
100 ml of fixing solution for 1 h. The fixer was replaced with fresh fixing solution
and incubated overnight on a horizontal shaker to remove SDS completely. The gels
were washed thrice with deionized water for 15 min each and stained with 100 ml of
phosphoprotein stain for 2 h in dark. The gels were transferred to destaining solution
and incubated for 15 min. Destaining was repeated twice more with fresh destaining
solution. The gels were washed with deionized water twice for 5 min each before
scanning. Scanning was carried out using Typhoon 9400 variable mode scanner
using the following parameters; ex 536 nm, em 580 nm, Long Pass filter and PMT
600 V. Green laser was used for scanning. After scanning, the gels were counter
stained with SYPRO®
Ruby total protein stain as described in section 13.3.
15. PEPTIDE MASS FINGERPRINTING BY MALDI-TOF MASS
SPECTROMETRY
Peptide mass fingerprinting by MALDI-TOF mass spectrometry was carried out as
described elsewhere [Gupta et al., 2007].
115
Extraction of gel plugs
The proteins to be identified by mass spectrometry were excised from the 2D gels,
manually, using a gel punch and transferred to low binding tubes. The gel plugs were
either processed immediately or stored at 4 ºC till the next step.
Destaining
The gel plugs were sliced into ~1mm cubes, using a sterile scalpel blade, to improve
the accessibility to the entrapped proteins. The gel pieces were washed in deionized
water thrice for 10 min each. Fifty microliters of destaining solution was added to
the gel pieces and incubated at room temperature for 15 min. This step was repeated
twice. The tubes were occasionally subjected to gentle mixing to aid destaining.
Tryptic digestion
The destained gel pieces were dehydrated by adding 100 l of 100% ACN and
incubated at room temperature for 15 min. The solution was discarded and the gel
pieces were dried completely under vacuum for 30 min. The dried gel pieces were
rehydrated with 5 l of trypsin (80 ng/ l) and incubated on ice for 30 min to achieve
complete absorption of the solution into the gel pieces. The rehydrated gel pieces
were overlaid with 20 l of 40 mM ammonium bicarbonate in 10% ACN and
incubated at 37 ºC for 16 h.
Extraction of digested peptides
The tryptic digested peptides were separated from the gel pieces in three steps. First,
the digestion mix was centrifuged at 12000 rpm for 30 sec and the supernatant was
transferred to a fresh tube. In the second step, 25 l extraction solution was added to
the gel pieces and sonicated for 3 min using a water bath sonicator at a frequency of
2200 MH. The tubes were centrifuged for 30 sec at 12000 rpm and incubated at
room temperature for 10 min. The supernatant was collected and added to the first
supernatant. To the gel pieces, 20 l of 100% ACN was added and vortexed
vigorously. The tubes were centrifuged for 30 sec at 12000 rpm and incubated at
room temperature for 10 min. The supernatant was collected and pooled with
previously collected supernatants. The extracted peptides were dried completely
116
under vacuum for 60-90 min. The dried peptides were either processed immediately
or stored at 4 ºC for later use.
Purification of peptides
The dried peptides were resuspended in 5 l of resuspension solution. The peptides
were desalted using C-18 reverse phase resin packed in a pipette tip (zip-tip). The tip
was fitted on a 10 l pipette and the plunger was brought to dead stop. First, the
micro column was wetted by aspirating and dispensing wetting solution for seven
times and then equilibrated in the equilibration solution in the same manner. The
peptides were aspirated and dispensed 10 times to bind to the C-18 resin. The salts
were washed off by aspirating and dispensing the wash solution 3 times. Finally, the
bound peptides were eluted into the elution solution.
Spotting
Each sample was spotted onto the target plate with both CHCA and DHB matrices.
Spotting with CHCA matrix was carried out by sandwich method as described
elsewhere [Beavis et al., 1992]. In this method, 0.5 l of the matrix was spotted on
the target plate and after drying, 0.5 l of the analyte was spotted onto this followed
by 0.5 l of the matrix to co-crystallize the peptides between two layers of matrix.
Dried droplet method was followed for spotting digested peptides in DHB matrix
[Karas and Hillenkamp, 1988]. In this method, 1 l of the matrix was mixed with
1 l of the analyte in a tube and 1 l of this mixture was spotted onto the MALDI
target plate. The spotting was done in duplicates using each matrix. Peptide mix was
spotted onto the target plate in respective matrices for calibration. After spotting, the
target plate was dried in plate drier for 20 min before loading into the mass
spectrometer.
Collection of spectrum
The target plate was allowed to remain in vacuum in mass spectrometer for 2-3 h
before starting the analysis. The laser power (described in arbitrary units) was
chosen based on the type of matrix used for spotting. i.e., approximately 65-75 was
used for CHCA spots whereas 100-120 was used for DHB spots. The spectrum
acquisition was calibrated using a mixture of standard peptides of known molecular
117
mass (bradykinin fragment (1-7) 757.39 Da; angiotensin II 1046.54 Da; P14R
1533.85 Da; ACTH fragment (18-39) 2465.19 Da) spotted along with the digested
peptides. A minimum of 200 profiles were collected per sample and monoisotopic
peaks were generated without applying peak smoothing function. The peak filter was
applied to exclude masses lower than 750 Da and higher than 3500 Da. The peak list
containing the m/z values of the peptides were exported for database searching.
Database searching
The m/z values were searched against Swiss-Prot or NCBInr database using
MASCOT (http://www.matrixscience.com) and/or MS-Fit (http://prospector.
ucsf.edu/) search algorithm for entries under the taxonomy Mus musculus.
Contaminating peaks from keratin and autolysed trypsin were removed before
database searching. A maximum of one missed cleavage and a variation in peptide
mass of 0.1-0.5 Da were tolerated. Carbamidomethyl modification of cysteine was
assigned as a fixed modification for peptides whereas oxidation of methionine,
propionamide modification of cysteine and conversion of glutamine to
pyro-glutamate were assigned as variable modifications. A protein was considered
identified when the probability based MOWSE score was above the threshold value
(p-value <0.05).
16. MS/MS ANALYSIS BY NANO LC-QTOF MASS SPECTROMETRY
Tryptic digestion of proteins and extraction of digested peptides were carried out as
described in section 15. For MS/MS analysis, the digested peptides were
reconstituted in 0.1% formic acid. The resuspension volume and injection volume
was decided based on the spot intensity obtained by IMP7 image analysis (Table
M.5). The peptides were separated using nano liquid chromatography system prior to
MS/MS analysis. Acclaim PepMap100 C-18 reverse phase capillary column was
used to separate the peptides under 80% acetonitrile and 0.1% formic acid gradient.
The separated peptides were sprayed at 220 nl/min flow rate for precursor ion scan
and the multi charged peptide ions were automatically selected for fragmentation.
Data analysis software 3.4 was used to process MS/MS spectra to generate data files
in .mgf format. These .mgf files were searched against Swiss-Prot or NCBInr
databases using MASCOT search algorithm for entries under the taxonomy
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Mus musculus. A variation of 0.05-0.2 Da was tolerated for the precursor ion,
whereas 0.2-0.6 Da was tolerated for fragment ion. One missed cleavage was
tolerated. Carbamidomethyl modification of cysteine was assigned as a fixed
modification whereas oxidation of methionine, propionamide modification of
cysteine and conversion of glutamine to pyro-glutamate were assigned as variable
modifications. A protein was considered identified when the probability based
MOWSE score was above the threshold value (p-value <0.05).
Table M.5: Peptide injection volume for LC MS/MS analysis
Intensity of the spot from
IMP7 analysis
Resuspension volume
( l)
Injection volume
( l)
Faint-20 10 8
20-50 20 8
50-80 20 5
80-100 20 3
120 20 1
17. WESTERN BLOTTING
Electroblotting
The protein samples were resolved using 13% polyacrylamide gel. The separated
proteins were blotted onto nitrocellulose membrane using semi-dry blotting method
as described by Ausubel et al., [1989] with minor modifications. The gel was
incubated in chilled transfer buffer [Towbin et al., 1979] for 10 min. A replicate gel
was taken for staining. Three small Whatman No. 2 filter papers and nitrocellulose
membrane having the same dimension as that of the gel and another three large
Whatman No. 2 filter papers, which are slightly larger than the gel dimension, were
soaked in chilled transfer buffer for 5 min. The large Whatman No. 2 filter papers
were arranged as one-on-top-of-the-other on the anode plate of the transfer apparatus
without trapping air bubbles. The nitrocellulose membrane soaked in transfer buffer
was placed on top of the Whatman papers and the gel was carefully placed over it
without trapping air bubbles. The small Whatman No. 2 filter papers were placed
over the transfer assembly and a glass rod was rolled over this to remove any trapped
119
air bubbles. The cathode plate was placed over the transfer assembly and the
electroblotting was carried out at 0.8 mA/cm2 for 1.5 h. After blotting, the
nitrocellulose membrane was transferred to a tray for immunodetection.
Immunodetection
The blot was incubated in blocking buffer at room temperature for 3 h. The blocked
membrane was rinsed with TBS and incubated in primary antibody solution (1:1000
dilution) at 4 ºC, overnight on a horizontal shaker. The blot was then rinsed thrice
with TBS-T for 5 min each and subsequently thrice with TBS for 5 min each to
remove the unbound antibodies. After rinsing, the blot was incubated in detection
antibody solution (1:2000 dilution) for 45 min in dark, at room temperature. The blot
was washed thrice with TBS-T for 5 min each, to remove the unbound detection
antibody. The blot was developed using hydrogen peroxide as substrate and DAB as
chromogen. The developer solution was poured off after developing and the
membrane was rinsed with deionized water several times.
18. IMAGE ANALYSIS
18.1 ImageMaster™ Platinum 2D version 7.0
The spot detection and matching for traditional 2D gels were carried out using
ImageMaster™ Platinum 2D software version 7.0. The digitized images were saved
in .tiff format in grayscale at 200 dpi. The images to be analyzed were imported to
the IMP7 image pool. The type of stain used for the detection of the spots was
specified for each gel. The images were cropped to remove the gel boundaries and to
select the areas of interest for comparison. Hierarchy matching method was
employed to match the gels. In this method, a new ‘workspace’ was created and
within which ‘match’ and ‘class’ folders were created. The replicate gels were
imported to separate subfolders. One of the gels was assigned as ‘reference’ gel in
each group. The spots were detected and to avoid the overlapping spots, pseudo
spots and gel artifacts the following detection parameters were specified. The
‘smooth’, a parameter which determines the number of times the software smoothens
the image before detection of spots so that it can distinguish overlapping spots, was
set between 9 and 12 for independent cases. The ‘saliency’, a parameter which
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measures the spot curvature, was set at a minimum of 10 for all the gels since the
real spots have high saliency and the non-protein artifacts will have smaller saliency
values. The ‘min area’ parameter which measures the area of the spot in the gel was
set between 6 to 9 for independent cases since the non-protein spots will contribute
only to a smaller area in the gel and can be excluded by this. After detection, the
spots were manually verified and edited, wherever necessary, using the edit spot
tool. A few well separated spots from the replicate gels were chosen as landmarks
before matching to enhance the match quality. The replicate gels from each group
were merged by selecting ‘merge matchset’ option. All the gels in the merged
matchset were matched to one another. The spot volume comparison for the matched
spots was exported as an excel work sheet. The statistical analysis was carried out by
importing the matched gels to the respective groups in ‘class’ folder. The spots
which showed a differential expression with an ANOVA value less than 0.05 were
considered significant.
18.2 DeCyder™ 2D version 7.0
18.2.1 Differential in gel analysis (DIA)
The 2D DIGE spot maps were edited using image editor tool to remove the gel
boundaries and the edited images were added to the project using image loader tool.
The DIGE gels were imported to DIA workspace for spot detection and intra-gel
spot matching. The spots present in each gel were detected and matched using
detection algorithm version 6.0 and upper detection limit was set at 2000 spots per
gel. Exclusion filters based on the peak height, volume, area and slope of the spots
detected were used to avoid the detection of artifacts as protein spots. The excluded
proteins were removed from the workspace and proteins showing a minimum of ±1.5
fold change in expression levels were imported into BVA module for inter-gel
matching and biological variation analysis.
18.2.2 Biological variation analysis (BVA)
The 2D DIGE gels for biological variation analysis were imported into the BVA
workspace after spot detection and intra-gel matching by DIA. The experimental
groups to be compared were assigned in the Spot map mode in BVA workspace. The
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control and experimental spot maps were segregated into separate groups and
internal standard gels were retained in a separate group named as Standard. The
master gel was selected based on the number of spots detected. Generally, the gel
with the highest number of spots detected was chosen as the master gel for inter-gel
matching. The spots detection was verified and the spots were edited, if necessary,
before matching. Inter-gel matching was carried out in the Match mode, where the
internal standard spot maps of gels were matched to the master gel. To enhance the
quality of matching a few proteins were assigned as ‘landmarks’ and they were
manually matched before matching the whole spots. Approximately 10 random spots
were chosen as landmarks from different parts of the spot map. The inter-gel
matching was improved by choosing the ‘warping’ function, where the gel image is
reshaped to fit the master gel image thereby correcting the distortions in the gel
images and building a geometrically corrected gel image. The matches were
manually assessed and the mismatches were corrected before carrying out statistical
analysis. Protein statistics were calculated in the Protein mode. The average fold
change in the normalized spot volume between control and experimental group was
calculated. Independent Student’s t-test was performed to assess the significance of
differential expression. The spots showing ±1.5 fold change with a p-value of <0.05
were considered significant and the remaining spots were excluded from the
workspace using protein filter. FDR correction was employed to minimize the risk of
false positive results. The differentially expressed proteins were identified by mass
spectrometry.
18.2.3 Extended data analysis
Extended data analysis on the random dataset of differentially expressed proteins
obtained by biological variation analysis was carried out to organize the
multidimensional dataset. The spots showing significant biological variation was
imported into the EDA workspace and it was assigned as the base set for further
calculations. The spots showing a significant difference in expression levels with
p<0.01 and of these, the spots present in more than 80% of the spot maps were
selected and this filtered set was stored as ‘T-test <0.01’ base set. Principal
component analysis was carried out on this new base set created. Two PCA
calculations were carried out, one on spot map versus proteins to check the quality of
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the control and experimental groups chosen for this study and the other on protein
versus spot map to organize the multidimensional data and to find the outliers, if
any. The outliers represent either mismatched spots or very highly differentially
expressed proteins. In order to verify this, outliers in the protein versus spot map
score plot were opened in BVA workspace and their inter-gel matching was
confirmed. The highly differentially expressed proteins were identified by mass
spectrometry and the expression pattern was validated using other methods.
Pattern analysis of the differentially expressed proteins was carried out by
hierarchial clustering and partition clustering methods. Two-way hierarchial
clustering was carried out using hierarchial clustering algorithm version 1.0. The
similarity in expression pattern was measured by Euclidean distance metrics and
distance between the two nodes in dendrogram was defined by average linkage
method. The protein spots were clustered based on their similarity in expression
profiles and the spot maps were clustered based on the expression pattern of proteins
to generate a heat map in Green/Red color scale. The heat map interval was set
between -1.5 to +1.5 to define the log standard abundance interval for the colors.
Partition cluster analysis was carried out using K-means algorithm version 1.0 for
non-hierarchial clustering. The number of clusters into which the differentially
expressed proteins are divided was defined based on the gap statistics. The
expression pattern of proteins in the clusters with the highest q-score was analyzed
further.
18.3 ImageQuant TL
Quantification of band volume from proteins resolved on 1D gels and immunoblots
was carried out using ImageQuant TL software, version 2003.02. The gels or blots
were scanned at 200 dpi using a desktop scanner. The images were saved in
grayscale in .tiff format and were imported into ImageQuant TL workspace. The
lanes were detected automatically and lane editing -moving, resizing or bending-
was done manually, wherever it was necessary. The background intensity from gel
or blot was subtracted employing ‘rolling ball’ method. The bands in gel or blot were
detected in automatic detection mode and the volume of bands in each lane was
calculated.
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19. QUANTIFICATION OF MOUSE HAPTOGLOBIN BY SANDWICH
ELISA
Preparation of standard graph
Quantification of mouse haptoglobin was carried out by sandwich ELISA. The
mouse haptoglobin calibrator (14.25 g/ml) was diluted in 1X sample diluent to
prepare the standard concentrations as described in table M.6. Hundred microliters
each of the mouse haptoglobin standards were added into the 96-well flat bottom
ELISA plates, pre-coated with capture antibody. A blank was prepared with 100 l
of 1X sample diluent. The microtitre plate was incubated at room temperature for
15 min to allow the binding of haptoglobin to capture antibody. The contents were
discarded and the wells were washed four times with wash solution using ELISA
plate washer. For each wash, 100 l of wash solution was dispensed to each well and
agitated for 5 sec before aspirating the contents. To the dry wells, 100 l each of 1X
enzyme-antibody conjugate (detection antibody) was added and plate was incubated
at room temperature for 15 min. The contents in the wells were discarded and wells
were washed as described before to remove the unbound detection antibodies.
Hundred microliters of chromogen-substrate solution was added to each wells and
the plate was incubated at room temperature in dark for 10 min. The color reaction
was stopped by the addition of 100 l of stop solution to each well. The absorbance
of the contents of each well was read at 450 nm using an ELISA plate reader. The
blank wells and standard wells were specified in the Microplate Manager®
software
version 5.2.1 (Bio-rad, USA) and the four-point logistic standard graph and curve
fitting equation was generated using default settings.
Optimization of sample dilution
The optimal sample dilution for estimation of haptoglobin concentration in control
and experimental mice plasma samples were identified as follows. Two
representative mice plasma samples from control and experimental groups were
taken. Each sample was diluted to 1:10000, 1:5000 and 1:2500 in 1X sample diluent
and each dilution was taken in duplicates to estimate the concentration of
haptoglobin by ELISA. The dilutions giving an absorbance value within the range of
haptoglobin standard curve were considered as optimal for subsequent experiments.
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Quantification of mouse plasma haptoglobin
Concentration of mice plasma haptoglobin was determined for 10 control and 20
M. leprae infected samples. Each sample was analyzed in duplicates and the mean
absorbance value was used to calculate the concentration of haptoglobin using 4-PL
curve fitting equation. The calculations were carried out using Microplate Manager®
software, version 5.2.1. The receiver operator characteristic was analyzed using Stata
software, version 5 (StataCorp LP, USA) using the default settings.
Table M.6: Serial dilution of mouse haptoglobin calibrator for standard curve
preparation
Standard Concentration (ng/ml) Volume of sample Volume of diluent ( l)
S7 125 5 l of calibrator 504
S6 62.5 250 l of S7 250
S5 31.25 250 l of S6 250
S4 15.6 250 l of S5 250
S3 7.8 250 l of S4 250
S2 3.9 250 l of S3 250
S1 1.95 250 l of S2 250
S0 0 Nil 500
20. ANALYSIS OF GLYCOSYLATION IN MOUSE HAPTOGLOBIN
The N-glycans attached to the mice plasma proteins were removed by PNGase F
enzyme treatment [Tarentino et al., 1985] by in-gel degylcosylation. Hundred
micrograms of mice plasma proteins were diluted to 40 l with deionized water and
boiled at 100 ºC for 10 min with 5 l of denaturation buffer to denature the proteins,
thereby making the glycosidic linkages accessible to enzyme active site. The
reaction mix was cooled to room temperature and 5 l of reaction buffer was added.
The samples were mixed well and divided equal volumes of the reaction mix into
two tubes. To one tube, 2 U of PNGase F enzyme (4 l) and to the other 4 l of
deionized water was added. Both the tubes were incubated at 37 ºC for 16 h. The
PNGase F minus reaction mix was included to overrule the interferences due to
protease activity in the reduction in molecular weight of glycosylated proteins. The
deglycosylation reaction was stopped by boiling at 100 ºC for 5 min. Ten microliters
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of the reaction mix was withdrawn from both the tubes and the proteins were
resolved using 13% polyacrylamide gels. The presence of haptoglobin in the
deglycoslated sample was detected by immunodetection using anti-haptoglobin
antibodies as described in section 17.
Recommended