Hi all,
I have repeated this western blot three times. Samples have been incubated with DTT 0.06 M at 99 oC for 5 min. The second time I prolonged the incubation to 10 min. The third time I doubled the DTT (0.12 M) and incubated 99 oC for 10 min, but my samples always look like this. Wells 1-2 belong to cell line 1, wells 3-4 belong to cell line 2, wells 5-6 belong to cell line 3, wells 7-8 belong to cell line 4. Do you have any suggestion?? Thank you!
Hi Martina!
The only suggestion that I have is to try to add b-mercaptoethanol to your samples together with 0.5M urea. Then you can try to boil your sample for 10' at 90°C instead of 99° for 5'. It could help to reduce your dimers. Try also to prepare a new batch of DTT stock solution. Sometimes it could oxidate.
Let me know if you will get any result. I hope I was useful.
Bests
Silvia
Alternative splicing variants, chemical crossbridges etc... without the POI and cell lines no one can say more. I guess your reduction is ok, at least an improper reduction could be seen if there is problem with it. if you guess membrane protein, lower the temp about 37 deg C.
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