I am struggling to get my LC3-I and LC3-II bands to separate on my western blot. I can see there is a difference in the protein levels but I'm not getting any distinction between the two.
I am currently collecting lysates with RIPA buffer and running them on a 15% SDS gel (I have also tried 12% and 18%) in Tris-Glycine buffer at 90V for up to 2h. I am then transferring to 0.2um PVDF membrane using a BioRad TransBlot Turbo.
I would like to be able to get a clear separation between these two bands so my analysis is a lot more reliable.
Hello Robert,
I just posted on the same issue. My bands aren't separating as well. If you find any better answers please let me know. Here is what I've found from trouble shooting.
1) amount of protease inhibitor may be to small resulting in instability.
2) transfer 80V for 1hr 30min in 10%methanol transfer buffer.
3) run 20% gel at 100V for 4 hrs
Let me know if any of these help.
Thanks,
-Konner W
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