MICRONUCLEUS ASSAY

The micronucleus test (m.t.) in vivo is a method devised primarily for screening chemicals for

chromosome-breaking effects.

PRINCIPLE:

In anaphase, acentric chromatid and chromosome fragments lag behind when the centric

elementsmove towards the spindle poles. After telophase the undamaged chromosomes, as well

as the centric fragments, give rise to regular daughter nuclei. The lagging elements are included

in the daughter cells, too, but a considerable proportion is transformed into one or several

secondary nuclei which are, as a rule, much smaller than the principal nucleus and are therefore

called micronuclei. Similar events occur if the functioning of the spindle apparatus is impaired,

e.g. under the influence of colchicine; in this event, however, the main nucleus is often replaced

by a whole group of small nuclei, which, in general, are considerably larger than typical

micronuclei.

In bonemarrow smears from mammals treated with chromosome-breaking agents, micronuclei

are found in numerous cell types, always provided that these cells have completed- under the

influence of the mutagen-one or a few mitoses. Micronuclei can be found e.g. in myeloblasts,

myelocytes and erythroblasts. A few hours after completion of the last mitosis, erythroblasts

expel their nucleus; for unknown reasons, micronuclei remain in the cytoplasm of the young

erythrocyte and there they are easily recognizable. As an ad-ditional advantage for the

micronucleus test, young erythrocytes stain differently from older forms. For the duration of

their adolescence, lasting approx. 24 h, they stain not red but bluish; to use a technical term: they

are polychromatic.

Micronuclei in polychromatic erythrocytes are characteristic and obtrusive elements which

can be scored by personnel without special training in cytogenetics. In hematological routine

these micronuclei have long been known as Howell-Jolly bodies.

Typically, micronuclei are round with a diameter of about 1/20 to 1/5 of an erythrocyte.

Protocol:

For peripheral blood MN Assay

Step 1: Preparation of smear on clean slide by cutting tail tip of animal by asceptic surgical

blade.

Step 2: Put smeared slide for air drying.

Step 3: Fixed in absolute methanol for 5min

Step 4: Put slide for air drying

Step 5: Stain with acridine orange (14mg/40ml) for 5min followed by three washing with

phosphate buffer pH 6.8.

It can be stained with Giemsa (20%).

Note: Acridine should be prepared in phosphate buffer pH 6.8.Keep the stained slide in dry and

dark condition.

Step 6: After drying slide view under 100X (oil emersion) fluroscent microscope in filter 3(blue

filter)

PROTOCOL FOR BONE MARROW MN ASSAY Step 1: Extraction of bone marrow from small animals. Before the animals are killed, a 5-ml

centrifuge tube is filled with fetal bovine serum for each individual.

Method for isolation of bone marrow from animal:

From the freshly killed animal both femora are removed in toto, which means that one is

cutting through pelvis and tibia. The bones are then freed from muscle by the use of gauze and

fingers. By gentle traction the distal epiphyseal portion is torn off together with the rest of the

tibia and the surrounding muscle. The proximal end of the femur is carefully shortened with

scissors until a small opening to the marrow canal becomes visible. With the needle of

appropriate size mounted, about 0.2 ml serum is pulled from the tube into a disposable plastic

syringe. Then the needle is inserted a few mm into the proximal part of the marrow canal which

is still closed at the distal end. Next, the femur is submerged completely in the serum and

squeezed against the tube to prevent the bone from slipping off the needle. Subsequently, the

marrow is aspirated; should the needle have become obstructed, the serum in the syringe is first

pressed out. After several gentle aspirations and flushings, the process is repeated from the distal

end of the femur. The bone marrow cells should get into the serum as a fine suspension and not

in the form of gross particles.

Step 2: The tube is centrifuged at 1000 rpm (4th unit) for 5 min. Discard the supernatant with a

Pasteur pipette. Pellet obtained is to be tapped

Step 3: A small drop of the viscous suspension is put on the end of a slide and spread thick

smear by pulling the material behind a glass slide held at an angle of 90о. The preparations are

then air dried

Step 4: Fix the smear in absolute methanol in copling jar for 5-10min and put it for air dry

Step 5: Stained with Giemsa for 10min diluted with distilled water 1: 6 ; rinse in distilled water

Step 6: Observation and scoring

Reference:

1. Mut. Res. 31 (1975) , The micronucleus test, W. Schmid 2. Mutation Research, 64 (1979) 45-46, Application of a simple giemsa-staining method in

the micronucleus test, B. Gollapudi and O.P. Kamra 3. OECD Guideline for the testing of chemicals ,474, Mammalian Erythrocyte MN Test.

CHROMOSOME ABERRATIONS ASSAY

PRINICIPLE: This test serves to detect structural chromosome aberrations, as may be induced

via DNA breaks by various types of mutagens. Such DNA breaks may either rejoin such that the

chromosome is restored to its original state, rejoin incorrectly or not rejoin at all. These last two

cases may be observable on microscopic preparations of metaphase cells. However, many of

these gross changes probably will not allow cell survival after division, but they serve as

indicators for the induction of smaller, not readily observable changes, which do allow cell

survival but may have deleterious consequences for the organism.

Basically this test is designed to determine whether a test material is clastogenic i.e. whether it

has the capacity to break chromosomes.Clastogenicity is an inmportant endpoint because it is

through breakage and inappropriate rejoining that certain oncogenes, such as myc, can be

activated and certain tumor suppressor genes,such as those causing retinoblastoma,can be

inactivated. To obtain a sufficient number of mitotic cells, an spindle inhibitor like may be added

shortly before fixation, to block cells in (pro)metaphase. An exogeneous metabolisation system,

like a liver microsome fraction, can also be added.

If the DNA damage occurred in GO or G1 phase and could not be repaired, chromosome type

aberrations will result: these involve both chromosome arms. Damaged that occurred in S or G2

phase causes chromatid type aberrations. Exceptions exist: chromatid type aberrations may be

induced if the DNA breaks result from errors in replication of otherwise damaged DNA.

Several types of aberrations can be distinguished:

• terminal deletions, give acentric fragments

• double minutes

• acentric rings

• centric rings

• inversions (can only be observed if banding techniques are used)

• reciprocal translocations (can only be observed if banding techniques are used)

• asymmetrical interchanges, giving dicentric chromosomes

Experimental protocol:

Step1: Before start to experiment Colchicine is to be administered i.p., 1.5-2hr before killing the animals (Colchicine solution 0.04%w/v , 20mg in 50ml distill water; Al foil coated, Bring the solution to 37оC at the time of administration)

Step 2: Remove the femur from the animal and flush with 0.56% w/v KCl 37оC solution and aspirate

Step 3: Incubate this extract in 0.56% w/v KCl in water bath at 37оC for 25 min

Step 4: Centifuge at 1000rpm(4th unit) for 10 min

Step 5: Discard the supernatant and tapped the pellet

Step 6: Treat the pellet with Cornoy’s fixative(1:3, GLACIAL ACETIC ACID: Methanol)The cells can be kept in the fixative till the preparation of slides.

Step 7: Centrifuge at 1000rpm for 10min

Step 8: Discard the supernatant, suspend the pellet in the carnoy’s fixative(white milky color suspension obtained)

Step 9: Using the pauster pipette drop the suspensions drops on the chilled (50% v/v) ethanol treated slides, from a height of 3-5ft and immediately burn the ethanol on it within 2-4 seconds

Step10: Dry at room temperature and stain using giemsa(1:6)

COMET ASSAY

Principle behind comet assay A single cell suspension is necessary for comet assay. The principle of the assay is based upon

the ability of denatured cleaved DNA fragments to migrate out of the cell under the influence of

an electric potential, whereas undamaged supercoiled DNA remains within the confines of the

cell membrane when a current is applied. Evaluation of the DNA “comet” tail shape and

migration pattern allows for assessment of DNA damage. The method involves the

immobilization of cells in a bed of low melting point agarose, followed by gentle cell lysis. After

treatment with alkali to denature the DNA and hydrolyze sites of damage, electrophoresis, and

staining of the sample with a fluorescent DNA intercalating dye, the sample is visualized under

the Carl zeiss microscope

There are two major versions of comet assay:

The first one is after Singh et al. (1988) who used alkaline lysing solution (pH~10) and alkaline

electrophoresis buffer (pH>13). This version is capable of detecting single- strand breaks and

alkali-labile lesions in the DNA of individual cells. This technique was called single cell gel

(SCGE) assay by Singh et al. (1998), although commonly referred to as comet assay. The second

version is after Olive and her coworkers who used mildly alkaline (pH 8) lysis followed either by

electrophoresis at neutral (Olive et al.1990) or alkaline (pH 12.3) conditions (Olive et al. 1990)

to detect double or single strand breaks respectively.

REAGENT PREPARATION LYSIS STOCK SOLUTION: Lysis Solution in per1000ml contains

2.5 M NaCl- 146.1g

100 mM EDTA- 37.2g

10 mM Trizma Base- 1.2g

Add ingredient about 700ml dW and begin stirring the mixture. Add 8gm NaOH and allow the

mixture to dissolve(about 20min). Adjustnthe pH to 10.0 using conc. HCl or NaOH q.s. to 890ml

with dW(the TritonX-100 and DMSO will increase the volume to correct amount) store at room

temperature.

NOTE: The purpose of the DMSO in the lysing solution is to scavenge radicals generated by the

iron released from Hemoglobin when blood or animal tissues are used.

Lysis solution prepared by adding 400µl Triton X-100, 4ml DMSO (It is required for samples containing heme, such as blood cells or tissue samples)and 35.6ml Lysis Stock Solution

NOTE: Chill at 2 - 8° C in refrigerator, for at least 20 minutes before use primarily to maintain the stability of the agarose gel. LOW MELTING AGAROSE: 0.5% and 1% LMA should be prepared in PBS. Loosen the cap

to allow for expansion then heat the bottle in microwave until the agarose is molten. Agarose

should be poured very slowly and need to be stirred continuously. Before going to start the

comet assay, Place the LMA in micromave for melting and then in a 37° C water bath

maintaining its tem.

RUNNING BUFFER (ELECTROPHORESIS BUFFER): 300mM/1mM EDTA

Prepare from Stock solution

1. 10N NaOH (200g in 500ml distill water)

2. 200mM EDTA (14.8g in 200ml distill water) - pH 10

Store both at room temperature

For 1lit: Use 5mlEDTA (200mM) +30ml NaOH(10N) q.s. distill water to 1000mL, mix well

(pH >13)

NEUTRALISING BUFFER

0.4M Tris-48.5g added to approx. 800ml distill water adjust pH to 7.5 with conc. (> 10M )HCl;

q.s. to 1000ml with distill water, store at room tem.

STAINING SOLUTION

1. Preparation of SYBR Green I Staining Solution. SYBR Green I 5 µL in TE Buffer (50ml), pH

7.5 (TE: 10 mM Tris, 1mM EDTA: pH 8.0,). For 100ml take 120mg Tris and EDTA 0.5ml

(from stock 200mM) . The diluted stock is stable for several weeks when stored at 2 - 8° C in the

dark.

2. Ethidium bromide (EtBr: 10X stock 20mg/ml). Add 10mg in 50ml distil water, store at room

temperature. For 1X stock- Mix 1ml with 9ml distil water

Preparation of Base Slides

1% NMA(500mg per 50 ml in distill water or milliQ water).Microwave until near boiling and the

agarose boiling.While NMA agarose is hot,dip frosted slide up to one third the frosted area and

gently remove.Wipe underside of slide to remove agarose and lay the slide on wiped table to dry

overnight. Mark the slides and store at room temperature. Avoid humidity conditions.

Isolation of lymphocytes for comet assay:

1.Take 20µl EDTA in microcentrifuge tube and blood sample(1.5ml)

2. In another centrifuge tube, taken 800 µl HBSS + 100 µl EDTA + 80 µl of blood sample(from

step 1). Added ficoll histopaque at the bottom of centrifuge tube.

3. Centrifuge at 2000rpm for 4min at 6 оC

4. Cut the microtip, take 150 µl of ring(hazy) lymphocytes layer

5. Add 800 µl PBS for washing

6. Centrifuge at 2500rpm for 5min at 4оC

7. Supernatant discarded, kept 50 µl supernatant in tube.

8. 0.5% LMPA and 1% LMPA heated in oven for 1min until clear

9. Remove and keep in water bath to maintained constant temp.

10. Put on the tile on ice

11. Start dry bath at tem 38.5 оC and placed empty centrifuge tube

12. For lymphocytes, add 1% 50 µl LMPA +50 µl isolated lymphocytes cell in microcentrifuge

tube

13. From this take 80 µl and spread on precoated slide (1%NMA), cover with

coverslip(24X60mm)

14. After 5-10min, remove coverslip

15. Add 100µl 1% LMA on the slide, cover with coverslip and put on chilled tile. After 5-10min,

remove coverslip.

16. Keep the slide on the lysis soln for 16-24hr/overnight

17. In case of tissue like liver, lung chopped in 800 µl HBSS+ 100 µl DMSO+ 100 µl EDTA and

put in ice.

18. Add 0.5%LMPA (95 µl) +5 µl sample. From this take 100 µl spread on glass slide cover with

coverslip and put it on chilled slide. After 5min put 3rd layer of 1% LMA and placed coverslip on

it and peform same step as for blood lymphocytes.

19. Wash the slide with chilled distil water for 5min

20. Incubate the slide with 1000ml Running buffer for 20min for DNA unwinding in

electrophoresis tank

21. Run electrophoresis at 300mA, 100W,30V/cm2 (length of 2 electrode 37X 0.8)=29.6V/cm2

for 30min,cover with black cloth. After 30min remove running buffer soln for thrice 5 min of

each

22. Add 1ml neutralising buffer soln for thrice 5min of each

23. Stain with 150-200µl SYBR GREEN per slide for 1hr cover with black cloth

24. Wash with chilled distil water for 5min

25. Then cover with 20X50mm coverslip

26. Keep slide in a box which saturated with dW soaked in a tissue paper, keep in 2-80C (Score

within 2 days)

For Lymphocytes- 0.7V/cm

For Bone marrow- 0.9 V/cm

SPERM COMET ASSAY

Step 1: Cut the epididymis and kept in a HBSS soln containing 10% EDTA, 10%DMSO for

15min and adjust the sperm no. 2-5x104 sperm/ml of soln.Take 5µl of sperm in 95 µl of 5%

LMA and layer it 80µl over base slides and perform the same as in normal comet upto layering

of agarose

Step 2: Lysis solution is same as normal comet assay as such, but also add 200 mg (for 40ml) of

dithiothreitol (DTT-40mM) in the coplin jar containing the lysis solution. Lysis is done for 24 h

at room temperature and not in freeze. DTT is added to break the disulphide bonds

Step 3: After lysis, slide were put in different coplin jar containing 0.5mg/ml proteinase K in a

buffer(NaCl 2.5M, Tris 5mMol, SDS 0.5%w/v) having pH 7.5-8.0 for 24hr for enzyme treatment

(here, proteinase K is added to the normal lysis solution not containing DTT). Proteinase K is

added to further break the structure of sperms which is very compact and highly condensed

Step 3: After enzyme treatment wash the slides with de-ionized water 3 times

Step 4: After washing put the slides in comet assay tank and incubate in the running buffer

(20min) and after incubation apply electrophoresis for 30min at 15V(0.6V/cm) 250mA

Step 5: Neutralize the slides with neutralization buffer (3 time 5 min each)

Step 6: After neutralization add 200 µl of SYBR Green on each slide and put on cover

slipElectrophoresis is carried out at 28 V and 250 mA

Composition of electrophoresis (running) buffer

NaCl 29.22 g (500 mM)

Trizma base 12.2 g (100mM)

EDTA 5 ml (1mM) stock 200mM

DMSO 2 ml (2%)

Dissolve in 900 ml distilled water, adjust pH 9.0 and then make up the volume upto 1000 ml.

MODIFIED COMET ASSAY FOR DETECTION OF OXIDIZES BASES WITH USE OF BACTERIAL REPAIR ENDONUCLEASE III AND FPG ENZYME

PREPARATION OF REAGENTS:

Enzyme Stock Solution

1 unit of enzyme is used for one slide.

Product: Sigma 10µg

230µg/ml of protein

10,000 unit mg/ml of protein

1mg/ml of protein- 10,000unit

0.230µg/ml of protein- 10,000unit

=10,000x0.230

=2300unit

230 µg-2300 unit

10 µg -100unit

Enzyme Reaction Buffer

10ml(1X) 100ml(10X)

HEPES(40mM) 104mg 1.040g

KCl(0.1M) 29.8mg 298mg

EDTA(0.5mM) 25µl(200mM stock) 250µl

BSA(0.2mg/ml BSA) 2mg 20mg

Adjust the pH 8.0 with KOH

Step 1: Isolation of lymphocytes and single cell method would remain same as in comet assay

Step 2: Embedding cells in agarose same as in normal comet assay

Step3: H2O2 treatment in case of standard curve preparation

Stock H2O2 is 8.8M, dilute 11.5µl in 1ml water-0.1M. Keep this as working stock and dilute and make con.in 10, 30, 100, 300 and 100µM of H2O2

Add 50 µl of above concentration of H2O2 cover with coverslip and have it on cold surface for 5min

0.1mM

10 µl-990 µl (PBS 1mM)

100+900 µl PBS 300+700 µl PBS

(100 µM) (300 µM)

100+900 µl PBS 100+900 µl PBS

(10 µM) (30 µM)

Step 4: Lysis was performed in lysis solution for 1hr at 40C

NOTE: If H2O2 has been used,any control slides must remain isolated i.e in a separate vessels

during lysis,otherwise strandbreak may occur in the control slide

Step 5: Enzyme Treatment

• Prepare 300ml of enzyme reaction buffer. Put aside 1ml for enzyme dilution

• Wash slides in 3changes of buffer(40C) in staining jar for 5min each

• Remove slides from last wash, and dab off excess liquid with tissue

Step 6: Alkaline treatment (40min) and Electrophoresis

• Electrophoresis solution should be cooled before use(0.3M NaOH 1mM EDTA)

• Gently place slide in the tank and poured electrophoresis buffer all slide should be dip for

40min, After 40min incubation, run the electrophoresis for 30min at 25V(0.8V/cm)

Step 7: Neutralisation

Thrice washing in duration of five minute each with neutralizing buffer I staining jar at 40C

Step 8: Staining

Stain with SYBR Green-I for 1hr

Table I. Enzymes to Study DNA

Enzymes DNA recognition site Action

Exonuclease III AP sites Nicks DNA backbone Proteinase K Protein DNA interactions Degrade protein Endonuclease III Thymine glycol,

dihdrothymine,dihydroxydihydrothymine.Uracil glycol,urea

Excises altered base and nicks DNA backbone

Mut M FPG

8 oxoguanine,8 –oxoadenine,foramidopyrimidines(FapyA, FapyG, methyl- fapy- guanine,aflatoxin B1-fapy-guanine), 5-hydroxy-cytosine,5-hydroxy-uracil,ring opened N-7guanine adducts(7 methylguanine), and AP sites

Excises altered base and nicks DNA backbone

HALO ASSAY

Same step should be performed as in comet assay except following:

• Immerse the slides in freshly prepared lysis solution (same composition as in comet lysis

solution) for 2hr at 40C

• Following lysis, incubate the slides with alkaline medium (0.3N NaOH) for 20min, Stain

using EtBr. Wash the slide with distil water for 5min and stained with EtBr

PROTOCOL FOR IMMUNOHISTOCHEMISTRY

PRINCIPLE

The fundamental concept behind IHC is the demonstration of antigens (Ag) within tissue

sections by means of specific antibodies (Abs).

Deparaffinize the sections with xylene 3 times for 3-5 min each.

Wash the slides with ethanol 3 times for 3-5 min each.

Wash the slides with PBS 3 times for 3-5 min each.

Incubate the slides with citrate buffer (pH 6.0 at 95-100°C) for 20 min in water bath for

antigen retrieval

Allow the slides to cool completely.

After antigen retrieval, wash the slides with PBS 3 times for 5 min each.

Remove the slides from PBS, wipe gently around each) section and cover tissue with 3%

H2O2 for 10 min at room temperature (to block endogenous peroxidase).

Wash the slides with PBS 3 times for 3-5 min each.

Remove the slides from PBS, wipe gently around each) section and cover tissue with

protein blocking agent (Ultra V block) for half an hour or 10 min accordingly.

Do not wash

Tip off blocking buffer, wipe gently around each section and cover tissues with primary

antibody (1~1000)

Incubate the sections overnight at 4°C.In case of GST-P incubate for 1hr at room temp

Wash the slides with PBS 3 times for 3-5 min each

Wipe gently around each section and cover tissues with polyvalent biotinylated antibody

Incubate for 1 hr at room temperature (place on a shaker)

Wash the slides with PBS 3 times for 3-5 min each

Wipe gently around each section and cover tissues with streptavidin peroxidise (it will

enhance the signal)

Incubate for 1/2 hr at room temperature

Wash the slides with PBS 3 times for 3-5 min each

Allow the sections to react with diaminobenzidine (DAB) for 10-15 min (it act as

chromogen)

Wash the slides in water for 5 min

Counterstain the sections with haematoxylin for 10 min (Harris haematoxylin)

Wash the slides with water 3 times for 3-5 min each.

Wash the slides with PBS 3 times for 3-5 min each.

Wash the slides with ethanol 3 times for 3-5 min each (for dehydration).

Wash the sections with xylene 3 times for 3-5 min each.

Mount the sections with DPX.

Preparation of citrate buffer:

• Tri-sodium citrate dehydrate (2.94 g)

• Distilled water (1000 ml)

• Adjust pH 6.0 with 1 N HCl and add 0.5 ml TWEEN 20

Preparation of DAB solution

DAB (7.5 mg) + 3 % H2O2 in 150 ml PBS. Add 1 drop of DAB solution to 1 ml diluents

Phosphate Buffer Saline (PBS)

NaCl 8.0g (0.137mM)

KCl 0.3g (2.7mM)

KH2PO4 0.24g (1.4mM)

Na2 HPO4 1.44g (0.01M)

• Distil water upto 1liter

• No need to adjust pH 7.4

TUNEL ASSAY PRINCIPLE:

During apoptosis endonucleases activated and cut the DNA results in the generation of free3`OH

groups at the end of these DNA fragments.The Fluorescein-Fragel TM dectection kit labels these

hydroxyl groups with fluorescein conjugated deoxynucelotides.This direct incorporation of

excitable bases simplifies the Fragel procedure by eliminating the need for secondary detection

steps resulting in rapid, sensitive, and specific staining of DNA fragments.

Step 1: Deparaffinization and rehydrartion

• Immerse slides in xylene for 5min at room tem.Repeat using fresh xylene for a second

5min for incubation

• Immerse in 100% ethanol for 5min.Repeat using fresh 100% ethanol for a second 5min

Slide + Specimen

Xylene(5min)

Xylene(5min)

100% alcohol(5min)

100% alcohol(5min)

90% alcohol(5min)

80% alcohol(5min)

70% alcohol(5min)

Rinse slide briefly with 1X TBS and carefully dry the glassslides around the specimen

NOTE: Do not let the specimen dry out during or between any step, if necessary cover or

immerse the specimen in 1X TBS to keep hydrated

Step 2: Permealization of specimen

Dilute 2mg/ml proteinase K 1:100 in 10mM Tris pH 8.0(mix 1µl of 2mg/ml proteinase K + 99µl

10mMTris per specimen)

Add 100µl of 20µg/ml proteinase K. Incubate at room tem. For 5min.

Rinse slide with 1XTBS

Gently tap of excess liquid and carefully dry the glass slide around the specimen

10mM Tris- 121.14MW, pH- 8.0

121.14g-1000ml-1M

1.2114g-1000ml-.01M ~10mM

0.1214g- 100ml ~10mM

TBS (20mM Tris pH 7.6, 140mM NaCl)

Tris

0.2428-10ml---20mM

X 5=1.2140 for 500M

NaCl

58.5 – 1000ml-1M

58.5x0.14-1000ml-0.140M

5.85x0.14-100ml-0.140M~140mM

0.8190g-100ml-140mM

X 5= 4.0950 in 500ml

Step 3: Equilibration and labelling Reaction

• Dilute 5xTdT equilibration buffer 1:5 with dH2O (mix 20µl 5X buffer with 80 µl dW)

• Cover the entire specimen with 100 µl of 1x TdT equilibration buffer. Incubate at room

tem for 30min,while preparing the labelling reaction mixture.

• Working TdT labelling reaction mixture, 57.0 µl Fluroscein –Fragel TdT labelling with 3

µl TdT enzymes

• Carefully blot the 1X equilibration buffer from the specimen taking care not to touch

specimen

• Immediately apply 60µl TdT labelling reaction mixture

• Cover the specimen with paraffin, place the slides in a humidified chamber and incubate

at 370C for 1-1.5hr

Step 3: Termination and Evaluation

• Remove paraffin,wash 3 times in TBS

• Wipe excess TBS from back of slide,mount a glass coverslip using florescein fragel

mounting media

• Total cell population visualized using DAPI

• Analysed labelled by using a std. fluorescein filter 465-495nm,store mounted sample at

40C

Thiobarbituric Acid Reactive Substances (TBARS)

Or Malonialdehyde (MDA) Assay

Principle Malondialdehyde (MDA) forms a 1:2 adduct with thiobarbituric acid. It can be measured by

fluorometry or spectrophotometry. Although this reaction has a much higher sensitivity when measured

via fluorometry. Mixture of thiobarbituric acid reactive substances (TBARS), including lipid

hydroperoxides and aldehydes, which increase as a result of oxidative stress. TBARS return to normal

levels over time, depending upon the presence of anti-oxidants.

A. Homogenization

• Take the tissue from the sacrificed animal (normal saline chilled)

• Weigh 1g tissue, transfer into test tube containing 4.5ml phosphate buffer + 3mM

EDTA(67.5µl 200mM)

• Transfer whole content in homogenisation tube

• Homogenise at 2000rpm and using 10 stroke (for liver)

B. Centifugation

• 700g for 10min(meanwhile switch on the watebath at 950C)

• Supernatant is taken for detection of lipid peroxidation

• 100 µl(supernatant) + 8.1% SDS(100 µl) + 20% CH3COOH (750 µl) +

0.8%TBA(750 µl) in glass test tube

• Volume make up to 20ml with dW(add 300 µl)

• Heated over waterbath at 950C for 60min(wrap the opening of test tube with Al

foil)

• Take out test tube stand and cool under tap water (pinkish color will develop)

• Centrifuge at 10,000rpm for 10min

C. Absorbance

Take 300 µl supernatant in microtiter plate, take blank and std soln (n=6)

Take absorbance at 532nm

Preparation of Std for MDA

Reagent 1,1,3,3 tetramethoxy propane(MW-164g/mol)

Take 17 µl std TMP + 983 µl dW(100 µM,1ml)

Serial dilution

(1nM,3 nM, 10 nM, 30 nM, 100 nM, 300 nM)

100 µM 1ml [A] Take 100 µl soln A,dilute to 1ml(add 900 µl dW)

10 µM 1ml [B] Take 100 µl soln B, dilute upto 1ml

1 µM 1ml [C]

Take 100µl soln [C] + 900 µl dW [C] Take 300µl soln [C] + 700 µl dW [C]

(0.1µM) 100nM 1ml [D] (0.3µM) 100nM 1ml [G]

100µl soln [D]+900µl dW 100µl soln [G]+900µl dW 10nM 1ml [E] 30nM 1ml [H]

100µl soln[E]+900µl dW 100µl soln [H]+900µl dW 1nM-1ml [F] 3nM-1ml [J]

Calculation of 17µl of std solution:

Required 100µM(1ml) of MW-164g/mole

164g in 1000ml – 1M

0.164g in 1000ml – 1mM

0.164 x10-3g in 1000ml - 1 µM

0.164 x 10-6g in 1ml - 1 µM

0.164 x 10-4g in 1ml – 100 µM

Vol. =mass/density

= 0.164x10-4/ 0.997(~1)

= 0.164x10-4ml

= 0.164x10-4 x 103µl

= 0.016µl

PROTEIN ESTIMATION (Lowry’s method)

Solution required:

Soln A- 2g sod. bicarbonate + 400mg sodium hydroxide in 100ml vol.flask

Soln B- 2% sod. tartarate soln

Soln C- 1% coppersulphate soln

Soln D- 96ml soln A +2ml soln B+ 2ml soln C

NOTE: Use soln D within 2hr of preparation

Standard: 1mg/ml Bovine Sreum Albumin(BSA)

Folin Reagent – 1:1 (Folin reagent:dW)

PROTOCOL: Volume and solution to be added(in following order)

Test tube

Distil Water Std Soln Test sample Soln D

Blank 500µl - - 2.5ml

Std 1 495µl 5µl - 2.5ml

Std 2 490µl 10µl - 2.5ml

Std 3 480µl 20µl - 2.5ml

Std 4 460µl 40µl - 2.5ml

Sample 495µl - 5µl 2.5ml

• Vortex all test tube after adding soln D and then incubate at 37оC in water bath for

10min

• After 10min,incubation,add folin reagent 250µl in each test tube

• Vortex tets tubes and incubate at 37оC for 30min

• Pour 300 µl from each test tube, duplicate in elisa plate at 660nm(absorbance)

GSH ASSAY

A. Homogenization

• Take the tissue from the sacrificed animal (normal saline chilled)

• Weigh 1g tissue, transfer into test tube containing 5ml PBS(pH-7.4) + 3mM

EDTA(75µl 200mM)

• Transfer whole content in homogenisation tube

• Homogenise at 2000rpm and using 10 stroke (for liver) B. Centifugation

• 700g for 10min(40C),Take 500 µl supernatant

• Add into eppendroff containing 500 µl 5% sulphosalicylic acid (freeze)

• White ppt will produced

• Vortex and keep it in ice for 30min

• Centrifugation at 10,000g for 10min

• Supernatant separated from pellet(can be stored in freezer,and perform rest steps

next day)

Ppn of Blank and Standard

One test tube- 500µl PB(7.4) Test tube

7 test tube- diff con of std GSH + PB(7.4) 400µl PB(7.4)+ 100 µl sample supernatant

Vortex

10min incubate in water bath(38оC)

Yellowish color will develop,Take absorbance(300µl in each well ) at 412nm

GSH standard preparation

6.14mg GSH 1ml PB(7.4) 20mM 40µl + 1960 µl(PB 7.4)

400µM final stock soln (2ml)

Ellman’s Reagent(DTNB- MW-396g/mole)

5,5’ – dithiobis(2-nitro-benzoic acid)

Prepare 0.1mM using PB(pH-8)

3.96g---------1000ml--------------0.01M

3.96mg-------1000ml--------------0.01mM

0.396g--------100ml----------------0.01mM In PB (pH-8)

3.96mg-------1000ml--------------0.01M

1.98mg-------50ml--------------0.1mM

No. STD. Std GSH

vol.(400µl) PB(7.4) Con.

Total vol.

(µl)

A Blank 0 µl 500 µl 0 500

B Std 5 µl 495 µl 4 500

C Std 10 µl 490 µl 8 500

D Std 20 µl 480 µl 16 500

E Std 40 µl 480 µl 32 500

F Std 80 µl 460 µl 64 500

G Std 160 µl 340 µl 128 500

H Std 320 µl 180 µl 256 500

HISTOPATHOLOGY

Take out the tissue from animal,put in formalin soln for fixing(change formalin if required)

Tissue processing

Treatment Duration Time

PBS 1hr 11.00 AM

PBS 1hr 12.00 PM

PBS 1hr 1.00 PM

70% alcohol 2hr 2.00 PM

80% alcohol 2hr 4.00 PM

90% alcohol 2hr 6.00 PM

100% alcohol 2 hr 8.00 PM

100% alcohol Overnight 10.00 PM

Xylene + Alcohol 2hr 9.00 AM

Xylene 1hr 11.00 PM

Paraffin 1hr 1.00 PM

Paraffin 1 hr 2.00 PM

NOTE:

• Excess time with xylene treatment makes tissue brittle so crtically follow the time

schedule

• Gradual increase in concentration of alcohol is required to show down the process of

shrinkage

• Switch on the paraffin embedder when 100% alcohol is changed by xylene alcohol

Staining Procedure

• Slides with section place in hot air oven to dry(40оC) at 15 min

• Treatment with Xylene(5min)

• Treatment with Xylene(5min)

• Treatment with 100% alcohol (3min)

• Treatment with hematoxylin(8min….15±5min) depends on the staining

• Treatment with 100% alcohol (2min)

• Treatment with 0.1% acid alcohol (30 sec)

• Treatment with distil water (5 min)

• Treatment with Scott Reagent (30 sec)

• Treatment with 2% Eosin solution (7-8min)

• Treatment with distil water (2 min)

• Treatment with 70% alcohol (2 min)

• Treatment with 80% alcohol(2min)

• Treatment with 100% alcohol (2min)

• Treatment with Xylene(5min)

• Air dry the slide after mounting it with DPX(40оC)(kept at 40 оC to attained adequate

viscous)

• Place coverslip and allow it to dry and observe under microscope

Reagent Used During Histopathology process

Phosphate Buffer Saline(PBS)

NaCl- 8.00g (0.137M)

KCl- 0.20g (2.7mM)

KH2PO4- 0.24g (1.4mM)

Na2H PO4- 1.44g (0.01M)

Add distil water to make up 1 liter, No need to adjust pH (7.4).

Microscope lens cleaning solution

o 30% Ethyl alcohol

o 70% diethyl ether

Buffered Neutral formal (10%) for 1 lit

Na2H PO4 -6.5g

NaH2 PO4- 3.5g

Formaldehyde(40%)-100ml

Distil water – 900ml

Mayer’s Albumin Solution: Take egg albumin from white of an egg and add equal quantity of

glycerol. Stirred it well (use magnetic stirrer). Add thymol crystals (2-4 small piece act as

preservative). Mix it properly.

Acid Alcohol: 2l ml distil water

+ 1ml con. HCl Take 15ml -35ml absolute alcohol (ethanol)

Proportion: Distil water: Acid : Alcohol(29:1:70)

Scott Reagent: 0.2%- Sodium bicarbonate

0.2%- Magnesium sulphate

Chromic acid mixture for cleaning glassware:

Take 200g sod.chromate. Add 100ml distil water (in ice bath). Now add 1500µl con. sulphuric

acid dropwise carefully (icebath)

Note: If a green color develops discard the mix in a sink with continuously flushing water

Preparation of Phoshphate Buffer

1. 0.2M dibasic NaPO4 3- : 1lt (X) For 500ml MW Na2HPO4.2H2O- 178.05 35.61g or Na2HPO4.7H2O - 268.07 53.95g or Na2HPO4.12H2O- 358.14 71.64g Make volume using distilled water upto 1lit.

2. 0.2M monobasic NaPO4 3- : 1lt (Y) For 500ml MW Na2HPO4.H2O - 138.05 27.6g

or NaH2PO4.2H2O - 156.03 31.21g Make volume using distilled water upto 1lit.

Working solution 0.1M-100ml Mix X ml of 0.2M dibasic sod. phosphate with Y ml of monobasic sod. phosphate. Dilute to 100ml with (dil 1:1) distil water, at 250C

pH ml of X ml of Y 5.8 4ml 46.0ml 6.0 6.15ml 43.75ml 6.2 9.25ml 40.75ml 6.4 13.25ml 36.75ml 6.6 18.75ml 21.25ml 6.8 24.50ml 25.50ml 7.0 30.5ml 19.50ml 7.2 36.0ml 14.00ml 7.4 40.5ml 9.50ml 7.6 43.5ml 6.5ml 7.8 45.75ml 4.25ml 8.0 47.35ml 2.65ml

DNA LADDER ASSAY

• Take 100-200 mg tissue in 1 ml LSB (Low Salt Buffer) and homogenate.

• Add 50 µl SDS (10%) and 10 µl proteinase K (1 mg/ml).

• Heat for 2 h at 56ºC in water bath or keep it overnight at room temperature (RT).

• Centrifuge at 10,000 g for 10 min at RT.

• Take supernatant and add an equal amount of phenol and chloroform (300 µl supernatant

+ 300 µl phenol + 300 µl chloroform).

• Shake it for 1 h on a shaker.

• Centrifuge at 10,000 g for 10 min at RT.

• Take supernatant and again add an equal amount of phenol and chloroform (300 µl

supernatant + 300 µl phenol + 300 µl chloroform).

• Centrifuge at 10,000 g for 10 min at RT.

• Again take supernatant carefully and add chilled ethanol (1:2) and 20 µl of 5 M NaCl.

• For precipitation of DNA, keep eppendorf at -20 ºC for 5-6 h or overnight.

• Carefully centrifuge eppendorf at 16,000 g for 20 min at 0 ºC and carefully remove the

ethanol and dry the pellet as much as possible.

• After pellet drying, dissolve it in 100 µl of TE (Tris/EDTA) buffer, add 10 µl of RNAse

and incubate at 37 ºC for 1 h in water bath.

• Take 5 µl sample in 1 ml TE buffer in cuvet and measure the absorbance in UV

spectrophotometer at 260 and 280 nm to find out DNA conc.

• DNA is ready for gel electrophoresis.

• Add 25 µl of sample buffer to DNA.

• Cast the gel with 1.5% high resolution agarose in 1X TE buffer.

• Allow the gel to solidify for 1 h, when it gets solidified, keep it in chamber and add the

running buffer approximately 2-3 mm above the gel and then add DNA sample into the

well.

• When the dye moves 2/3rd part of the gel, stop electrophoresis and visualize the gel in

transluminator.

Reagents composition:

(1) DNA ladder marker.

40 µl step (ladder) std. + 10 µl sample buffer.

(2) TBE (Tris/Borate/EDTA) buffer

1000 ml 500 ml

Tris base 445 mM 53.84 g 26.92 g

Boric acid 445 mM 27.51 g 13.75 g

Tri sodium EDTA 10 mM 3.72 g 1.86 g

Do not adjust Ph, it should be 8.0 at RT.

(3) Low Salt Buffer (LSB)

2000 ml 1000ml 100 ml

Tris base 12.1 g 6.05 g 605 mg

NaCl 18 g 9 g 900 mg

Adjust pH 7.4 then make up the volume up to the required amount (2000 or 1000 or 100 ml) and

store at 4ºC.

DNA FRAGMENTATION ASSAY

• Take 100-200 mg tissue in 1 ml lysis buffer without adding triton-X, then homogenate

• Transfer 800 µl of cell suspension in an eppendorf and add 700 µl ice-cold lysis buffer,

vortex and allow lysis to proceed for 15-30 min at 4ºC.

• Centrifuge at 15,000 g for 15 min. at 4ºC.

• Transfer supernatant to a small test tube and add 1.5 ml of 10% TCA (trichloroacetic

acid).

• Add 650 µl of 5% TCA to a small test tube and add 1.5 ml of 10% TCA.

• Precipitate the samples overnight or for ≥ 4 h at 4ºC.

• Centrifuge the supernatant at 2500 g for 10 min at RT.

• Remove the supernatant afte centrifugation and add 650 µl of 5% TCA to the pellet.

• Prepare 2 blank eppendorfs with 650 µl of 5% TCA and treat similarly for the remaining

steps.

• Boil both the sets of eppendorfs for 15 min at 100ºC.

• Cool to RT, centrifuge at 2500 g for 5 min at RT.

• Transfer 0.5 ml of supernatant from both eppendorfs and add 1 ml of diphenylamine

reagent.

• Incubate for ≥ 4 h at 37 ºC in water bath (6 h).

• Read absorbance at 600 nm in spectrophotometer.

• Express results as the % of DNA fragmented. % of DNA fragmented = [abs. of the

supernatant/( abs. of the supernatant+ abs. of the pellet)]*100

Reagents: Diphenylamine reagent

• 100 ml glacial acetic acid

• 1.5 g diphenylamine

• 1.5 ml conc. Sulphuric acid

• 0.5 ml 16 mg/ml acetaldehyde

Prepare acetaldehyde stock in ultrapure water, store in a dark flask up to 1 month at 4ºC.

Lysis buffer

5 Mm Tris HCl, pH 8/20 mM EDTA/0.5%v/v Triton-X 100.

WESTERN BLOTTING

PRINCIPLE:  Western blotting identifies with specific antibodies proteins that have been

separated from one another according to their size by gel electrophoresis. The blot is a

membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed

next to the membrane and application of an electrical current inducesthe proteins in the gel to

move to the membrane where they adhere. The membrane is then a replica of the gel’s protein

pattern, and is subsequently stained with an antibody. Protocol:

A. Sample preparation

1. Lysis buffers

2. Protease and phosphatase inhibitors

3. Preparation of lysate from tissues

4. Determination of protein concentration

5. Preparation of samples for loading into gels

B. Electrophoresis

1. Preparation of PAGE gels

2. Positive controls

3. Molecular weight markers

4. Loading samples and running the gel

5. Use of loading controls

C. Transfer of proteins and staining (Western blotting)

1. Visualization of proteins in gels

2. Transfer

3. Visualization of proteins in membranes: Ponceau Red

4. Blocking the membrane

5. Incubation with the primary antibody

6. Incubation with the secondary antibody

7. Development methods

REAGENT AND BUFFER PREPARATION 1. Seperating Buffer (Buffer A, pH-8.8) 1.875M Tris (Trizma) 22.7g Trizma Dissolve it in 80ml distil water

                                                                                                Adjust the pH with con.HCl to 8.8

    Make up volume upto 100ml

2. Stacking Buffer (Buffer B, pH-6.8)

0.6M Tris (Trizma) 7.3g Trizma Dissolve it in 80ml distil water

Adjust the pH with con.HCl to 6.8

Make up volume upto 100ml

3. Stock Acyrlamide Solution (30%)

Acrylamide -30g

Bisacrylamide-800g

• Dissolve it in 70ml of DW by stirring (1-2 hr) and make up the volume upto 100ml.

• Kept it in refrigerator at 4ºC.

NOTE- Acrylamide is a neurotoxin so care must be taken during its weighing and handling.

4. Ammonium Persulphate(APS) 10%

100mg APS dissolved in 1ml DW.

NOTE- Prepare it fresh during the casting of gel.

5. Running (Electrophoresis) Buffer

5X 1X

Trizma 15g 3g

Glycine 72g 14.4g

SDS 5g 1g

1lit 1lit

6. Sample Buffer (pH 6.8)

10ml 50ml

0.6M Tris HCl (pH 6.8) 1ml 5ml

SDS 0.1g 0.5g

Sucrose or glycerol 1g or 1ml 5g or 5ml

Β Mercaptoethanol 0.05ml 0.25ml

0.5% Bromophenol blue 1ml 5ml

D.W. to make up volume q.s. 10ml 50ml (q.s.)

7. Seperating Gel

10% 12% 14%

5ml 10ml 5ml 10ml 5ml 10ml

30% Acrlyamide

1.7ml 3.4ml 2.0ml 4.0ml 2.4ml 4.8ml

Seperating Buffer pH 6.8

1.0ml 2.0ml 1.0ml 2.0ml 1.0ml 2.0ml

Distil water 2.25ml 4.5ml 1.9ml 3.8ml 1.6ml 3.2ml

10% SDS 50µl 100µl 50µl 100µl  50µl  100µl 

APS (10% w/v)

30µl  50µl  30µl  50µl  30µl  50µl 

TEMED 7µl  7µl  7µl  7µl  7µl  7µl 

8. Stacking Gel 5ml 10ml

Distil water 3.75ml 7.5ml

Stacking buffer (pH6.8) 0.5ml 1.0ml

30% Acrylamide 0.67ml 1.34ml

10% SDS 50µl 100 µl

10%w/v APS 30µl 50µl

TEMED 7µl 7µl

9. Staining Solution For 200ml

Methanol- 100ml

DW- 80ml Mix and add 200mg of Coomassie brilliant blue dye

Add 20ml Glacial acetic acid

Stir for 1-2hr and filter

0.3% Brialliant blue + 50% methanol + 75%GAA + 42.5% DW

10. Destaining Solution

7.5% GAA + 50% methanol and make up the volume up to 1 lit with DW.

Or Methanol-20ml

GAA -14ml

DW- 166ml

200ml

11. Transfer Buffer (Towbin buffer)

500ml

Tris(25mM) 1.5g

Glycine(192mM) 7.2g

Methanol(20%v/v) 100ml

SDS(0.1%) 0.5g

DW(q.s.) 500ml

NOTE- SDS facilitate the elution of high molecular weight proteins

12. TBS and TBST

Prepare stock solution of Tris and NaCl separately.

Tris (2M) – 242.28g in 1000ml

NaCl (5M) – 292.5g in 1000ml

Take 10ml of Tris (2M) & 30ml of NaCl (5M) and make up the volume upto 900ml adjust pH

with con. HCl at 7.2 and then make up the volume upto 1000ml.

For TBST preparations takes TBS solution (500ml) and 0.5ml of Tween-20 and stir it on

magnetic stirrer.

13. Stripping Buffer (500ml pH-6.8)

Tris 50mM – 3.78g

SDS 2% - 10g

B- mercaptoethanol 100mM- 3.9ml

Dissolve 3.78g Tris in 400ml water and adjust the pH 6.8.Then add 10g SDS and dissolve then

adjust the volume upto 500ml. Don’t add B-mercaptoethanol.

NOTE- For stripping of one blot take 50ml of solution and then add 390ml B-mercaptoethanol

and then incubate the blot at 60ºC for 30minute with gentle shaking.

14. Homogenising Buffer

Tris(2M) 100ml

EDTA(0.5M) 1ml

NaCl(5M) 0.2ml

NP-40 1ml

SDS 100mg

Make up volume upto 100ml. Maintain pH 7.4-7.5.

NOTE- In the above homogenizing buffer add PMSF, NaVO3,NaF and protease inhibitor just before the homogenization.

5ml

NaF 500µl

NaVO3 5µl

PMSF 50µl

Protease inhibitor 10µl

PMSF- (100mM) Stock FW-174.19

174.19g in 1000ml-1M

174.19mg in 1ml -1M

174.19mg in 10ml-0.1M

174mg in 10ml isoprapanol (100mM Stock)

STEP INVOLVED IN WESTERN BLOTTING

Sample preparation • Homogenise tissue in bufferA(1:10)

• Filter it through muslin cloth

• Layer it over buffer B(20ml)

• Centrifuge at 3000rpm for 10min

• Dicard supernatant, suspend pellet in 5ml buffer and triton –X (10%) 500µl with

vortexing

• Layer it over buffer B(10ml) centifuge at 3000rpm for 10min at 40C

• Discard supernatant

• Suspend pellet in 5ml buffer A

• Layer it over buffer B,centrifuge at 3000pm

• Dicard supernatant

• Resulting pellet contain crude nuclei

• Store at -800C (if donot want to proceed the some day)

• Pellet containing crude nuclei

• Dissolve pellet in RIPA buffer or,LSB containing protein inhibitor(sodium

orthovandate,PMSF)

• Sonicate 3times for 10sec,perform protein estimation

• Add sample buffer(1:1)

• Heat in boiling water for 3-4min

Buffer A ppn (pH 7.4) 12% sucrose+10mMTris + 10mMEDTA +10mM NaCl +1mMNaB +1mMPMSF 100ml 500ml Sucrose(12%) 12g 60g Tris 0.5ml 2.5ml NaCl(10mM) 200 µl 1000 µl NaB(1 mM) 100 µl 500 µl PMSF(1mM) 1000 µl 5ml

Buffer B ppn(pH 7.4) 100ml 500ml Sucrose(15%) 15g 75g EDTA(10mM) 2ml 10ml Tris(10mM) 0.5ml 2.5ml NaCl(10mM) 200 µl 1000 µl NaB(1 mM) 100 µl 500 µl PMSF(1mM) 1000 µl 5ml or Sample preparation To prepare samples for running on a gel, cells and tissues need to be lysed to release the proteins

of interest. This solubilizes the proteins so they can migrate individually through a separating

gel.There are different homogenizing buffer or lysis buffer chosing accordingly their interest of

protein.

1. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to

prevent degradation by proteases.

2. Place the tissue in round-bottom microfuge tubes or Eppendorf tubes .Store samples at -80°C

for later use or keep on ice for immediate homogenization.For a ~5 mg piece of tissue, add ~300

μl lysis buffer rapidly to the tube, homogenize with an electrichomogenizer, rinse the blade twice

with another 2x300 μl lysis buffer, then maintain constant agitation for 2hours at 4°C (e.g place

on an orbital shaker in the fridge). Volumes of lysis buffer must be determined in relation to the

amount of tissue present (protein extract should not be too diluted to avoid loss of protein and

large volumes of samples to be loaded onto gels. The minimum concentration is 0.1 mg/ml,

optimal concentration is 1-5 mg/ml).

3. Centrifuge for 20 min at 12000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from

the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice;

discard the pellet.

The buffer (with inhibitors) should be ice-cold prior to homogenization. Determination of protein concentration Perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a

frequently-used protein standard.

Once determined the concentration of each sample, freeze them at -20°C or -80°C for

later use or prepare for immunoprecipitation or for loading onto a gel.

Preparation of samples for loading into gels (denatured sample) Denatured sample, for this use a loading/sample buffer with the anionic denaturing detergent

sodium dodecyl sulfate (SDS), and boil the mixture at 95-100°C for 5 minutes.

SDS denatures proteins by “wrapping around” the polypeptide backbone.

Mercaptoethanol used to reduce the disulphide bond

Glycerol added to increase the density of sample and maintain the sample at the bottom

of the well

Bromophenol used to enable a visualization of migration of protein

NOTE- During protein sample treatment the sample should be mixed by vortexing before and after the heating

stepfor best resolution.

Preparation of PAGE (PolyAcrylamide Gel Electrophoresis) gels 1. Preparation of stacking and seperating gel as described above. According to the protein size

gel percentage varies. Add the 3ml of separating gel in casting chamber. After half an hour add

stacking gel. Place the comb and leave for 10-15 min to cast. NOTE: The smaller the size of the protein of interest, the higher the percentage of mono/bis. The bigger the size of

the protein of interest, the lower the percentage of mono/bis.

2. Now remove the comb and put the gel loading guide so that sample can easily be loaded by

special gel loading tips

3. The gel submerged in migration/running/electrophoresis buffer

4. Run the gel for the recommended time.For stacking gel at 75V and after running over stacking

gel double the voltage for separating buffer.

When the dye molecule (the “migration front”) reaches the bottom of the gel, the power turned

off. Proteins slowly elute from the gel at this point, so do not store the gel; proceed immediately

to transfer.

Loading controls such as beta actin are required to check that the lanes in gel have been

evenly loaded with sample.

Transfer of proteins and staining (Western blotting) 1. Transfer the gel on membrane, it can be semidry and wet transfer. For both kinds of transfer,

the membrane should be placed next to the gel. The two are sandwiched between absorbent

materials, and the sandwich is clamped between solid supports to maintain tight contact between

the gel and membrane (sponge/paper/gel/membrane/paper/sponge).

2. Two types of membranes are available: nitrocellulose and PVDF (positively-charged nylon).

The choice is personal and both work very well. PVDF membranes require careful pre-treatment:

cut the membrane to the appropriate size then soak it in methanol for 1-2 min. Incubate in ice

cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold

transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of

transfer.

NOTE-The balance of SDS and methanol in the transfer buffer, protein size, and gel percentage

can affect transfer efficiency. The following modifications will encourage efficient transfer.

.

Visualization of proteins in membranes: ponceau red 1. To check for success of transfer, wash the membrane in TBST (for a TBST recipe, see below).

Dilute the stock Ponceau Red 1:10. The stock is made of 2% Ponceau S in 30% trichloroacetic

acid and 30% sulfosalicylic acid.

2. Incubate on an agitator for 5 min.

3. Wash extensively in water until the water is clear and the protein bands are well-defined.

4. The membrane may be destained completely by repeated washing in TBST or water. When

using a PVDF membrane, re-activate the membrane with methanol then wash again in TBST.

Blocking the membrane 1. Blocking the membrane prevents non-specific background binding of the primary and/or

secondaryantibodies to the membrane (which has a high capacity at binding proteins and

therefore antibodies).

2. To prepare a 5% milk or BSA solution, weigh 5 g per 100 ml of Tris Buffer Saline Tween20

(TBST) buffer. Mix well and filter. Failure to filter can lead to “spotting” where tiny dark grains

will contaminate the blot during development.

3. Incubate for 1 hour at 4°C under agitation. Rinse for 5 seconds in TBST after the incubation.

Incubation with the primary antibody

• Incubate the antibody in blocking buffer, Incubation time can vary between a few hours

and overnight (rarely more than 18 hours), and is dependent on the binding affinity of the

antibody for the protein and the abundance of protein.

• Incubation temperature preferably cold. If incubating in blocking buffer overnight, it is

imperative to incubate at 4°C or contamination will incur and thus destruction of the

protein (especially phospho groups). Agitation of the antibody is recommended to enable

adequate homogenous covering of the membrane and prevent uneven binding.

Incubation with secondary antibody • Wash the membrane several times in TBST while agitating, 5 minutes or more per wash,

to remove residual primary antibody.

• Incubation Buffer and Dilution: Dilute the antibody in TBST at the suggested dilution.

If the datasheet does nothave a recommended dilution, try a range of dilutions (1:1000-

1:20,000) and optimize the dilution according to the results. Too much antibody will

result in non-specific bands. You may incubate the secondary antibody (and primary

antibody) in blocking buffer, but a reduction in background may come at the cost of a

weaker specific signal, presumably because the blocking protein hinders binding of the

antibody to the target protein.

• Incubation Time and Temperature: 1-2 hours, room temperature, with agitation.

Development methods HRP-conjugated antibodies ,camera detects the chemiluminescence emanating from the

membrane, transforming the signal into a digital image for rapid analysis with software provided

with the detection machine.