Western blot complete protocol


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Western blot complete protocol

  1. 1. Accessories for western blotting 1. Gel Accessories a) Ammonium per sulfate b) TRIS PH 6.8 (1M) c) TRIS PH 8.8 (1.5) d) Temed e) Acrlamide/Bis (30:80) 2. Protein marker 3. 4X buffer 4. Running buffer 5. Transfer buffer 6. 20X Ponceau Stain 7. 5% Non-Fat Milk. 8. Primary Antibody 9. Secondary Antibody 10. 10X TBS 11. 1X TBST 12. ECL-Kit (Enhanced Chemiluminescence-Kit) 1.a Ammonium per sulfate Take 0.14g/140mg Ammonium per sulfate and dissolve it in distilled water, making final volume 1ml. 1.b TRIS PH 6.8 (1M) First prepare 0.2M EDTA and, 10% SDS. 0.2M EDTA (200 ml) = Take 11.6896g of EDTA and dissolve in distilled water by magnetic stirring, making final volume 200ml. SDS 10% (250 ml) = Take 25g SDS; dissolve in 100ml of distilled water by magnetic stirring. Color becomes transparent upon final dissolution. Then make final volume 250ml with the distilled water. Now go further to prepare 1M, 200ml TRIS. So take 24.228gm of TRIS. Dissolve in 100ml of water. Adjust PH 6.8. Add 8ml of 0.2M EDTA and 10% SDS each. Make final volume 200ml with distilled water. So it will be 1M, 200ml TRIS, PH 6.8, having 8ml 0.2M EDTA and 10% SDS each. 1.c TRIS PH 8.8 (1.5M) Prepare 0.2M EDTA and, 10% SDS as above.
  2. 2. Now go further to prepare 1.5M, 250ml TRIS. Take 45.4275gm of TRIS. Dissolve in 100ml of water. Adjust PH 8.8. Add 10ml of 0.2M EDTA and 10% SDS each. Make final volume 250ml with distilled water. So it will be 1.5M, 250ml TRIS, PH 8.8, having 10ml 0.2M EDTA and 10% SDS each. 1.d Temed We buy it ready made from company. 1.e Acrylamide/Bis (30 : 0.8) We make 1 liter solution. For this we take 300g= Acrylamide, and 8g= Bis. Dissolve in 500ml of distilled water. Then make final volume 1000ml. Filter the solution in clean bench and store at 4°C. 2. Protein Marker Std. Protein Marker:1X buffer 1:50 (e.g 50ul & 950ul) 3. 4X buffer 1M Tris (PH 6.8)---------- 1.25ml Glycerol-------------------- 4ml β- mercaptoethanol---- 2ml 1% BPB-------------------- 0.25ml D. Water------------------ 2.5ml SDS------------------------- 0.8g -------------------------------------------------------------- Total----------------------- 10ml 4. Running buffer We prepare 5X Tris running buffer, 2 liter. Tris= 30g, Dissolve in 500 ml of distilled water. Then make the volume 2 liters SDS= 10g, with the distilled water. Glycine= 144g 5. Transfer buffer We make 1 liter of transfer buffer. It contains: 100mM CAPS PH 11= 100ml, Methanol= 100ml, Distilled water= 800ml. Whereas,
  3. 3. 100mM CAPS= 22.132g CAPS/liter of distilled water, as; 1000mM/1M CAPS= 221.32g/ liter of distilled water. 1M= 1000mM 100mM= 1000mM/10, SO, 221.32/10=22.132g 6. 20X Ponceau Staining Solution 2% (W/V) Ponceau S in 30% Tricholoroacetic acid and 30% sulfosalicylic acid in distilled water. We can buy it ready made. When using in western blot, we make it 1X by taking 1ml 20X ponceau stain soln. and adding 19ml distilled water to it or half of these i.e 500ul and 9.5ml. 7. 5% Non-Fat Milk Non-Fat Milk= 2g powder. Dissolve in 1X TBS, making final volume 40ml with 1X TBS. 8. Primary Antibody We make it by 1:1000, primary antibody and 1X TBS respectively. So; 1X TBS---------------------- 10ml 1° Anb---------------------- 10ul 2% Sodium azide-------- 20ul 9. Secondary Antibody We make it by 1:2000, secondary antibody and 1X TBS respectively. So; 1X TBS---------------------- 20ml 1° Anb---------------------- 10ul No sodium azide in this case. 10. 10X TBS Tris= 48.4g, Nacl= 160g, Distilled water q.s to make 2 liters. When using, we make it 1X by taking 100ml 10X TBS and 900ml distilled water.
  4. 4. 11. 1X TBST 1 liter 1X TBS, and add 1ml tweens-20, or half of these quantities. 12. ECL- Kit (Enhanced Chemiluminescence-Kit) It contains two solutions. Mix both of these 1:1 and vortex. Ready to apply.
  5. 5. Western Blotting 1) Spray 70% ethanol on the glasses to be used in making assembly. Then clean with tissue paper. 2) Set the assembly and fix it in the stand. Add water into it and wait for 5-10 min for checking either water is leaking or not. 3) When water is not leaking, start making gel according to the specifications given in table below. We usually make 10% separation gel and 5% stacking gel. Table. 1 Gel Composition Separation Gel Gel %age Acrylamide/Bis 30:0.8 Tris-Base PH 8.8 D.Water APS Temed 7% (1) 7% (2) 7% (4) 1.75ml 1.88ml 3.88ml 3.75ul 75ul 3.5ml 3.75ml 7.75ml 7.5ul 150ul 7ml 7.5ml 15.5ml 15ul 300ul 7.5% (2) 7.5% (4) 3.75ml 3.75ml 7.5ml 7.5ul 150ul 7.5ml 7.5ml 15ml 15ul 300ul 8% (1) 8% (2) 8% (3) 8% (4) 2ml 1.88ml 3.63ml 4ul 75ul 4ml 3.75ml 7.25ml 7.5ul 150ul 6ml 5.63ml 10.88ml 12ul 225ul 8ml 7.5ml 14.5ml 15ul 300ul 10% (1) 10% (2) 10% (3) 10% (4) 2.38ml 1.88ml 3.13ml 4ul 75ul 4.75ml 3.75ml 6.25ml 7.5ul 150ul 7.12ml 5.63ml 9.38ml 12ul 225ul 10ml 7.5ml 12.5ml 15ul 300ul 12% (1) 12% (2) 12% (4) 3ml 1.88ml 2.63ml 3.75ul 75ul 6ml 3.75ml 5.25ml 7.5ul 150ul 12ml 7.5ml 10.5ml 15ul 300ul 12.5% (2) 12.5% (4) 6.25ml 3.75ml 5ml 7.5ul 150ul 12.5ml 7.5ml 10ml 15ul 300ul 15% (2) 7.5ml 3.75ml 3.75ml 7.5ul 150ul Stacking Gel Gel %age Acrylamide/Bis 30:0.8 Tris-Base PH 6.8 D.Water APS Temed 3% (2) 1.5ml 3.8ml 8.8ml 15ul 300ul 4.6% (2) 4.6% (4) 4.6% (6) 0.58ml 0.95ml 2ml 4ul 75ul 1.15ml 1.9ml 4ml 7.5ul 150ul 2.3ml 3.8ml 8ml 15ul 300ul 5% (2) 5% (4) 1.2ml 2ml 4.3ml 7.5ul 150ul 2.4ml 4ml 8.6ml 15ul 300ul 4) First make separation gel. For this, in a corning tube, take: (i) Acrylamide (ii) Tris-base PH 8.8 (iii) Distilled water (iv) Ammonium per sulfate, and (v) Temed. (vi) Now vortex for few seconds. Before adding TEMED, make assembly ready for pouring gel into it with the help of 5ml Tip.
  6. 6. (vii) Pour the gel solution into the assembly up to the Mark present in the stand. Add Iso- propanol/water in the remaining space for making the upper edge of the gel smooth. Wait for 20-30 min. In this time gel will be formed. 5) Now remove the water/iso propanol from the assembly, dry the upper empty part of gel with absorbent paper and insert the appropriate comb just above the upper edge of the gel. Start making 5% stacking gel in a corning tube. Add: (i) Acrylamide (ii) Tris-base PH 6.8 (iii) Distilled water (iv) Ammonium per sulfate (v) TEMED. (vi) Vortex for few seconds and transfer very quickly to the assembly in between the wells formed over the separating gel by comb with the help of 1ml tip. Take intensive care that no water bubble is entrapped. Wait 10-15 min for gel formation. 6) During the formation of stacking gel, prepare the sample for loading. We mix protein sample with 4X buffer and distilled water. And quantity of sample is taken according to the Std. Curve formula, e.g. y= 0.0648x + 0.0026(Used in MS Excel). Sample Absorbance 2ug 1ug 20ug Load H2O 4X buffer Total Control 0.247 3.83 1.91 10.47 4.53 5 20 3 hours 0.251 3.89 1.95 10.25 4.75 5 20 Absorbance= Y values (0.247, 0.251) 2ug(X value from y= 0.0648x + 0.0026) = Qty of sample used in analysis by UV spectrophotometer. 1ug= 2ug/2. 20ug Load= Desirable & variable load. 20/1.91= Loading Qty. divided by first value in 1ug column. H2O= Subtract the highest value in 20ug column from 15. (Total volume you make). 4X buffer= 1/3 of total volume (15/3=5). Total= Add last three columns. 7) After combining protein sample, distilled water and 4X buffer, give a flash on centrifugator and then keep the sample for 5 minutes in boiled water. 8) Again give a flash in the centrifugator. Now remove the comb, wash the wells with distilled water twice, dry with absorbent paper and load the sample in the following manner: 9) Now in running apparatus, add 1X running buffer, and start running at 90 volts for 2-2.5 hours. Take care of right positions of the electrodes while supplying power, red on red and black on black. Check visually that running buffer has started upward movement after getting power supply. 10) After completion of running time, transfer the gel to Nitrocellulose membrane. For this take Transfer cassette and keep: 1X running buffer 10-20ul Protein Marker 10-20ul Sample Sample Sample Sample Sample 1X running buffer 10-20ul 1X running buffer 10-20ul
  7. 7. Sponge Filter paper Gel NC membrane Filter paper Sponge NC paper size= 9 x 6 inch 11) Close the transfer cassette, add transfer buffer in transfer apparatus, insert the transfer cassette into it and start the transferring process. Apply 90 volts for 25 minutes, and then change the positions of cassettes and run for 25 minutes again. Take care of red and black sides. 12) On completion of transferring time, remove the NC paper from cassette, and add to 1X Ponceau stain solution. It will make protein bands visible. After that wash the NC paper with distilled water and cut it according to the molecular weight of proteins you need. Protein marker will be showing you the range of molecular weight. After making the NC strips, wash for 5 minutes in distilled water and then keep in 5% Non-fat milk at -20°C for 2 hours. This step is called blocking. 13) After blocking step, put the strips in appropriate primary antibody for 12-16 hrs at -20°C. 14) Wash the strips with TBST for 5, 5, 10, 10, and 10 minutes. 15) Apply appropriate secondary antibody for 2 hours keeping at -20°C. 16) Wash the strips with TBST for 5, 5, 10, 10, and 10 minutes. 17) Dry the strips by keeping it on tissue paper and then fix these in X-ray cassette, pour solution from ECL-Kit (1:1), wait for 2 minutes so that the strips properly absorb the solution, then fix the X-ray in this cassette in the dark room. (We usually fix 2 X-ray at first, then after taking result of first one, we fix 1 another X-ray and takes its result within 24 hours). 18) After fixing the X-ray wait for 30-40 minutes. Then develop the X-ray and take the result. Properly label you results.