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 90. 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 37C at the time of administration) Step 2: Remove the femur from the animal and flush with 0.56% w/v KCl 37C solution and aspirate Step 3: Incubate this extract in 0.56% w/v KCl in water bath at 37C 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 Cornoys 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 carnoys 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 400l 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 L 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 20l EDTA in microcentrifuge tube and blood sample(1.5ml) 2. In another centrifuge tube, taken 800 l HBSS + 100 l EDTA + 80 l of blood sample(from step 1). Added ficoll histopaque at the bottom of centrifuge tube. 3. Centrifuge at 2000rpm for 4min at 6 C 4. Cut the microtip, take 150 l of ring(hazy) lymphocytes layer 5. Add 800 l PBS for washing 6. Centrifuge at 2500rpm for 5min at 4C 7. Supernatant discarded, kept 50 l 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 C and placed empty centrifuge tube 12. For lymphocytes, add 1% 50 l LMPA +50 l isolated lymphocytes cell in microcentrifuge tube 13. From this take 80 l and spread on precoated slide (1%NMA), cover with coverslip(24X60mm) 14. After 5-10min, remove coverslip 15. Add 100l 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 l HBSS+ 100 l DMSO+ 100 l EDTA and put in ice. 18. Add 0.5%LMPA (95 l) +5 l sample. From this take 100 l 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 150-200l 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 5l of sperm in 95 l of 5% LMA and layer it 80l 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 l 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 Trizma base EDTA DMSO 29.22 g (500 mM) 12.2 g (100mM) 5 ml (1mM) 2 ml (2%) stock 200mM

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 10g 230g/ml of protein 10,000 unit mg/ml of protein 1mg/ml of protein- 10,000unit 0.230g/ml of protein- 10,000unit =10,000x0.230 =2300unit 230 g-2300 unit 10 g -100unit Enzyme Reaction Buffer 10ml(1X) HEPES(40mM) KCl(0.1M) EDTA(0.5mM) BSA(0.2mg/ml BSA) 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 11.5l in 1ml water-0.1M. Keep this as working stock and dilute and make con.in 10, 30, 100, 300 and 100M of H2O2 104mg 29.8mg 25l(200mM stock) 2mg 100ml(10X) 1.040g 298mg 250l 20mg

Add 50 l of above concentration of H2O2 cover with coverslip and have it on cold surface for 5min 0.1mM 10 l-990 l (PBS 1mM)

100+900 l PBS (100 M)

300+700 l PBS (300 M)

100+900 l PBS (10 M)

100+900 l PBS (30 M)

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 Exonuclease III Proteinase K Endonuclease III Mut M FPG DNA recognition site AP sites Protein DNA interactions Thymine glycol, dihdrothymine,dihydroxydihydrothymine. Uracil glycol,urea 8 oxoguanine,8 oxoadenine,foramidopyrimidines(FapyA, FapyG, methyl- fapy- guanine,aflatoxin B1-fapy-guanine), 5-hydroxy-cytosine,5hydroxy-uracil,ring opened N-7guanine adducts(7 methylguanine), and AP sites Action Nicks DNA backbone Degrade protein Excises altered base and nicks DNA backbone 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 95-100C) 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 4C.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 KCl KH2PO4 Na2 HPO4 8.0g (0.137mM) 0.3g (2.7mM) 0.24g (1.4mM) 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 1l of 2mg/ml proteinase K + 99l 10mMTris per specimen) Add 100l of 20g/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 20l 5X buffer with 80 l dW) Cover the entire specimen with 100 l of 1x TdT equilibration buffer. Incubate at room tem for 30min,while preparing the labelling reaction mixture. Working TdT labelling reaction mixture, 57.0 l Fluroscein Fragel TdT labelling with 3 l TdT enzymes Carefully blot the 1X equilibration buffer from the specimen taking care not to touch specimen Immediately apply 60l 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 40 C

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(67.5l 200mM) Transfer whole content in homogenisation tube Homogenise at 2000rpm and using 10 stroke (for liver) 700g for 10min(meanwhile switch on the watebath at 950C) Supernatant is taken for detection of lipid peroxidation 100 l(supernatant) + 8.1% SDS(100 l) + 20% CH3COOH (750 l) + 0.8%TBA(750 l) in glass test tube Volume make up to 20ml with dW(add 300 l) 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

B. Centifugation

C. Absorbance Take 300 l 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 l std TMP + 983 l dW(100 M,1ml) Serial dilution (1nM,3 nM, 10 nM, 30 nM, 100 nM, 300 nM) 100 M 1ml [A] Take 100 l soln A,dilute to 1ml(add 900 l dW) 10 M 1ml [B] Take 100 l soln B, dilute upto 1ml 1 M 1ml [C] Take 100l soln [C] + 900 l dW [C] (0.1M) 100nM 1ml [D] 10nM 1ml [E] 100l soln[E]+900l dW 1nM-1ml [F] Calculation of 17l of std solution: Required 100M(1ml) of MW-164g/mole 164g in 1000ml 1M 0.164g in 1000ml 1mM 0.164 x10-3g in 1000ml - 1 M 0.164 x 10-6g in 1ml - 1 M 0.164 x 10-4g in 1ml 100 M Vol. =mass/density = 0.164x10-4/ 0.997(~1) = 0.164x10-4ml = 0.164x10-4 x 103l = 0.016l Take 300l soln [C] + 700 l dW [C] (0.3M) 100nM 1ml [G] 100l soln [G]+900l dW 30nM 1ml [H] 100l soln [H]+900l dW 3nM-1ml [J]

100l soln [D]+900l dW

PROTEIN ESTIMATION (Lowrys 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 Blank Std 1 Std 2 Std 3 Std 4 Sample 500l 495l 490l 480l 460l 495l Std Soln 5l 10l 20l 40l Test sample 5l Soln D 2.5ml 2.5ml 2.5ml 2.5ml 2.5ml 2.5ml

Vortex all test tube after adding soln D and then incubate at 37C in water bath for 10min After 10min,incubation,add folin reagent 250l in each test tube Vortex tets tubes and incubate at 37C for 30min Pour 300 l 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(75l 200mM) Transfer whole content in homogenisation tube Homogenise at 2000rpm and using 10 stroke (for liver) 700g for 10min(40C),Take 500 l supernatant Add into eppendroff containing 500 l 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- 500l PB(7.4) 7 test tube- diff con of std GSH + PB(7.4) Vortex 10min incubate in water bath(38C) Yellowish color will develop,Take absorbance(300l in each well ) at 412nm Test tube 400l PB(7.4)+ 100 l sample supernatant

B. Centifugation

GSH standard preparation 6.14mg GSH 1ml PB(7.4) 20mM 40l + 1960 l(PB 7.4)

400M final stock soln (2ml)

Ellmans 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 3.96mg-------1000ml--------------0.01M 1.98mg-------50ml--------------0.1mM No. A B C D E F G H STD. Blank Std Std Std Std Std Std Std Std GSH vol.(400l) 0 l 5 l 10 l 20 l 40 l 80 l 160 l 320 l PB(7.4) 500 l 495 l 490 l 480 l 480 l 460 l 340 l 180 l Con. 0 4 8 16 32 64 128 256 Total vol. (l) 500 500 500 500 500 500 500 500 In PB (pH-8)

HISTOPATHOLOGY Take out the tissue from animal,put in formalin soln for fixing(change formalin if required) Tissue processing Treatment PBS PBS PBS 70% alcohol 80% alcohol 90% alcohol 100% alcohol 100% alcohol Xylene + Alcohol Xylene Paraffin Paraffin Duration 1hr 1hr 1hr 2hr 2hr 2hr 2 hr Overnight 2hr 1hr 1hr 1 hr Time 11.00 AM 12.00 PM 1.00 PM 2.00 PM 4.00 PM 6.00 PM 8.00 PM 10.00 PM 9.00 AM 11.00 PM 1.00 PM 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(40C) at 15 min Treatment with Xylene(5min) Treatment with Xylene(5min) Treatment with 100% alcohol (3min)

Treatment with hematoxylin(8min.155min) 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(40C)(kept at 40 C to attained adequate viscous) Place coverslip and allow it to dry and observe under microscope

Reagent Used During Histopathology process Phosphate Buffer Saline(PBS) NaClKClKH2PO4Na2H PO48.00g (0.137M) 0.20g (2.7mM) 0.24g (1.4mM) 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

Mayers 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: Proportion: Scott Reagent: 2l ml distil water + 1ml con. HCl Take 15ml -35ml absolute alcohol (ethanol) Distil water: Acid : Alcohol(29:1:70) 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 1500l 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) MW Na2HPO4.2H2O- 178.05 or Na2HPO4.7H2O - 268.07 or Na2HPO4.12H2O- 358.14 Make volume using distilled water upto 1lit. 2. 0.2M monobasic NaPO4 3- : 1lt (Y) MW or Na2HPO4.H2O - 138.05 NaH2PO4.2H2O - 156.03 For 500ml 35.61g 53.95g 71.64g

For 500ml 27.6g 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 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0

ml of X 4ml 6.15ml 9.25ml 13.25ml 18.75ml 24.50ml 30.5ml 36.0ml 40.5ml 43.5ml 45.75ml 47.35ml

ml of Y 46.0ml 43.75ml 40.75ml 36.75ml 21.25ml 25.50ml 19.50ml 14.00ml 9.50ml 6.5ml 4.25ml 2.65ml

DNA LADDER ASSAY

Take 100-200 mg tissue in 1 ml LSB (Low Salt Buffer) and homogenate. Add 50 l SDS (10%) and 10 l proteinase K (1 mg/ml). Heat for 2 h at 56C 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 l supernatant + 300 l phenol + 300 l 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 l supernatant + 300 l phenol + 300 l chloroform). Centrifuge at 10,000 g for 10 min at RT. Again take supernatant carefully and add chilled ethanol (1:2) and 20 l of 5 M NaCl. For precipitation of DNA, keep eppendorf at -20 C for 5-6 h or overnight. Carefully centrifuge eppendorf at 16,000 g for 20 min at 0 C and carefully remove the ethanol and dry the pellet as much as possible. After pellet drying, dissolve it in 100 l of TE (Tris/EDTA) buffer, add 10 l of RNAse and incubate at 37 C for 1 h in water bath. Take 5 l 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 l 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 l step (ladder) std. + 10 l sample buffer. (2) TBE (Tris/Borate/EDTA) buffer 1000 ml Tris base Boric acid Tri sodium EDTA 445 mM 445 mM 10 mM 53.84 g 27.51 g 3.72 g 500 ml 26.92 g 13.75 g 1.86 g

Do not adjust Ph, it should be 8.0 at RT. (3) Low Salt Buffer (LSB) 2000 ml Tris base NaCl 12.1 g 18 g 1000ml 6.05 g 9g 100 ml 605 mg 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 4C.

DNA FRAGMENTATION ASSAY

Take 100-200 mg tissue in 1 ml lysis buffer without adding triton-X, then homogenate Transfer 800 l of cell suspension in an eppendorf and add 700 l ice-cold lysis buffer, vortex and allow lysis to proceed for 15-30 min at 4C. Centrifuge at 15,000 g for 15 min. at 4C. Transfer supernatant to a small test tube and add 1.5 ml of 10% TCA (trichloroacetic acid). Add 650 l 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 4C. Centrifuge the supernatant at 2500 g for 10 min at RT. Remove the supernatant afte centrifugation and add 650 l of 5% TCA to the pellet. Prepare 2 blank eppendorfs with 650 l of 5% TCA and treat similarly for the remaining steps. Boil both the sets of eppendorfs for 15 min at 100C. 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 C 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 4C. 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 gels 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 4C.

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 Trizma Glycine SDS 15g 72g 5g 1lit 6. Sample Buffer (pH 6.8) 10ml 0.6M Tris HCl (pH 6.8) SDS Sucrose or glycerol Mercaptoethanol 0.5% Bromophenol blue D.W. to make up volume q.s. 7. Seperating Gel 10% 5ml 30% Acrlyamide Seperating Buffer pH 6.8 Distil water 10% SDS APS (10% w/v) TEMED 1.7ml 1.0ml 2.25ml 50l 30l 7l 10ml 3.4ml 2.0ml 4.5ml 100l 50l 7l 5ml 2.0ml 1.0ml 1.9ml 50l 30l 7l 12% 10ml 4.0ml 2.0ml 3.8ml 100l 50l 7l 5ml 2.4ml 1.0ml 1.6ml 50l 30l 7l 14% 10ml 4.8ml 2.0ml 3.2ml 100l 50l 7l 1ml 0.1g 1g or 1ml 0.05ml 1ml 10ml 50ml 5ml 0.5g 5g or 5ml 0.25ml 5ml 50ml (q.s.) 1X 3g 14.4g 1g 1lit

8. Stacking Gel Distil water Stacking buffer (pH6.8) 30% Acrylamide 10% SDS 10%w/v APS TEMED

5ml 3.75ml 0.5ml 0.67ml 50l 30l 7l

10ml 7.5ml 1.0ml 1.34ml 100 l 50l 7l

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) Glycine(192mM) Methanol(20%v/v) SDS(0.1%) DW(q.s.) 1.5g 7.2g 100ml 0.5g 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. Dont 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 60C for 30minute with gentle shaking.

14. Homogenising Buffer Tris(2M) EDTA(0.5M) NaCl(5M) NP-40 SDS 100ml 1ml 0.2ml 1ml 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 NaVO3 PMSF Protease inhibitor 500l 5l 50l 10l

PMSF- (100mM) Stock 174.19g in 1000ml-1M 174.19mg in 1ml -1M 174.19mg in 10ml-0.1M

FW-174.19

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%) 500l 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 Sucrose(12%) Tris NaCl(10mM) NaB(1 mM) PMSF(1mM) 100ml 12g 0.5ml 200 l 100 l 1000 l 500ml 60g 2.5ml 1000 l 500 l 5ml

Buffer B ppn(pH 7.4) Sucrose(15%) EDTA(10mM) Tris(10mM) NaCl(10mM) NaB(1 mM) PMSF(1mM) 100ml 15g 2ml 0.5ml 200 l 100 l 1000 l 500ml 75g 10ml 2.5ml 1000 l 500 l 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 -80C for later use or keep on ice for immediate homogenization.For a ~5 mg piece of tissue, add ~300 l lysis buffer rapidly to the tube, homogenize with an electrichomogenizer, rinse the blade twice with another 2x300 l lysis buffer, then maintain constant agitation for 2hours at 4C (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 4C 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 -20C or -80C 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 95-100C 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 4C 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 4C 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 (1:10001:20,000) 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.