For doing western blots, you need to have your samples (proteins )ready!
The followings are the basic steps:
1. clean the glass plates and spacers well.
2. make your stacking gel and resolving gel.
3. don't forget to put comb in the stacking gel.
4. load your samples.
5. set the voltage around 100-150V and run the gel till the dye reaches the end of your gel.
6. take out gel very gently, shrink it in blotting buffer.
7. get your transfer cassette ready. make a sandwich in the following order: the sponge, whatman paper, gel, nitrocellulose membrane, whatman paper and sponge. Make sure no bubbles get trapped in between the layers. Then close the cassette tightly. Put the sandwich-cassette in blotting buffer in blotting/transfer apparatus. orient in proper direction(- or +).
8. Run the transfer for 90 mins at 100V.
9. Get the nitrocellulose out gently, mark the sides.
10. Stain with fast green for 5 mins.
11. Destain for 15 mins.
12. Wash with TBS for 3 times, 10 mins each.
13. Wash with TTBS for 3 times, 10mins each.
14. Block in blocking buffer for 30mins.
15. Prepare your primary antibody dilution in blocking buffer.
16. Incubate the membrane in the primary antibody for overnight/16hrs at 4 C
17. Next day, remove primary antibody.
18. 2 times wash with TBS followed by 2 times wash with TTBS.
19. Incubate in secondary antibody.
20. Wash 2 times with TTBS followed by 2 times wash with TBS.
21. Get reactive reagent which will react with secondary antibody to give out the signal.
22. Develop/expose the signal on photographic films in dark room.
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