HOST-PATHOGEN INTERACTIONS IN LEPROSY …shodhganga.inflibnet.ac.in/bitstream/10603/124814/8/08...60 Water quality Deionized water of resistivity 18.2 m :/cm was used for preparation

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


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


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


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


Stored at room temperature.

Reagent B

Copper sulfate - 1.0 g



Reagent C

Sodium potassium tartrate - 1.0 g



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


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



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)


65

Cathode buffer

Sodium hydroxide - 0.4 g (0.1 M)


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


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



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



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


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


water before use.

Neutralization buffer

Tris HCl - 0.1 M

pH 8.0


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



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


before use.

Neutralization buffer

Tris HCl - 0.1 M

pH 8.0


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


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



10% APS

Ammonium persulfate - 0.1 g

69


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


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


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


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


10% APS - 0.05 ml

TEMED - 0.01 ml

1% Bromophenol blue

Bromophenol blue - 100.0 mg

Tris base - 60.0 mg


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)


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


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


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


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


1.5 M Tris-Cl pH 8.8 - 125.0 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%)


1.5 M Tris-Cl pH 8.8 - 62.5 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%)


1.5 M Tris-Cl pH 8.8 - 62.5 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%)


1.5 M Tris-Cl pH 8.8 - 62.5 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%)


1.5 M Tris-Cl pH 8.8 - 62.5 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


Labeling buffer

Urea - 4.2 g (7 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


Prepared fresh before use.

50 mM Sodium hydroxide

Sodium hydroxide - 20.0 mg


SOLUTIONS FOR COLLIODAL COOMASSIE BLUE STAINING

Fixing solution

Methanol - 500.0 ml

Acetic acid - 100.0 ml


Colloidal coomassie blue stain


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



Rinsing solution

Ethanol - 300.0 ml


Sensitizing solution

Sodium thiosulfate - 0.2 g

Deionized water - 1.0 liter


Stain

Silver nitrate - 1.0 g

Chilled deionized water - 1.0 liter


Developing solution



Just before use, added,

37% Formaldehyde - 400.0 µl

Stop solution



77

B) Glutaraldehyde sensitizer method

Fixing solution

Ethanol - 400.0 ml




Ethanol - 300.0 ml

Sodium thiosulfate - 2.0 g

Sodium acetate - 68.0 g


Just before use added,

25 % Glutaraldehyde - 20.0 ml

Stain



37 % Formaldehyde - 0.4 ml

Formaldehyde was added at the time of use.

Developing solution



37% Formaldehyde - 0.3 ml

Formaldehyde was added at the time of use.

Stop solution

EDTA - 18.6 g


C) Ammoniacal silver method

Fixing solution

Ethanol - 400.0 ml



78

Rinsing solution

Ethanol - 50.0 ml




Sodium acetate - 41.0 g

25% Glutaraldehyde - 40.0 ml


0.1% Napthalenedisulfonic acid

NDS - 0.5 g


Stain

Solution A



Solution B

10 N Sodium hydroxide - 2.0 ml

Ammonia - 13.2 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


At the time of use added,

37% Formaldehyde - 2.7 ml

Stop solution

Tris base - 50.0 g



79

SOLUTIONS FOR PHOSPHOPROTEIN STAINING

Fixer

Methanol - 50.0 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


SOLUTIONS FOR SYPRO® RUBY STAINING

Fixer

Methanol - 100.0 ml



Stain

SYPRO®

Ruby stain was diluted 1:1 with deionized water and used for staining.

Destaining solution

Methanol - 10.0 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


80


B) Solutions for tryptic digestion

100 mM Ammonium bicarbonate in 10% ACN


Acetonitrile - 100.0 l



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


Overlay solution

Ammonium bicarbonate - 3.2 mg (40 mM)

Acetonitrile - 100.0 l (10%)



C) Extraction solution

5% Trifluoroacetic acid - 20.0 l (0.1%)

100% Acetonitrile - 600.0 l (60%)



D) Resuspension solution



81



E) Solutions for zip-tip purification

Wetting and elution solution




Prepared fresh and aliquoted 50 l each for wetting and 5 l each for elution.

Equilibration and washing solution



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%)



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%)


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


ACTH stock (100 pmol/ l)

ACTH (18-39) - 10 nmol

0.1% TFA - 100.0 l


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%)


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


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


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



Tris buffered saline-Tween (TBS-T)




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



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


Stored at -20 ºC as 100 l aliquots.

Sample diluent

5X concentrate - 10.0 ml


Wash solution

20X concentrate - 25.0 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



Reaction buffer




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


3 112 1.62x106 MDT Balb/C /3 months 5x103







10 119 4.85x106 Untreated 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

























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.

104

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 3 1000 V Gradient 1 h

Step 4 5000 V Gradient 1.5 h


Active rehydration; 4-7 L, 11cm IPG strips

0 V Rehydration 1 h






106

Active rehydration; 3-10 NL, 18 cm IPG strips

0 V Rehydration 1 h




Step 4 8000 V Gradient 1.5 h


Active rehydration; 4-7 L, 18 cm IPG strips

0 V Rehydration 1 h






Passive rehydration; 3-10 NL, 7 cm IPG strips





Passive rehydration; 3-10 NL, 18 cm IPG strips





Cup loading; 3-10 NL, 13 cm IPG strips





107

Cup loading; 4-7 L, 13 cm IPG strips





Cup loading; 3-10 NL, 18 cm IPG strips





Cup loading; 4-7 L, 18 cm IPG strips





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

108

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.

109

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.

112

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.

113

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].

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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

120

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

121

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.

123

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.

124

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

125

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.

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HOST-PATHOGEN INTERACTIONS IN LEPROSY …shodhganga.inflibnet.ac.in/bitstream/10603/124814/8/08...60 Water quality Deionized water of resistivity 18.2 m :/cm was used for preparation