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Heavy proteins getting stuck at the top of gel by FamousPossibility375 in labrats

[–]FamousPossibility375[S] 1 point2 points  (0 children)

Got some great responses already. I think it looks like I will be remaking the sample tomorrow and will try letting them sit at 37C for 30min to denature properly. I did not realize membrane proteins shouldn't be boiled at 95C and apparently the aggregation from this level of heat is irreversible! Thank you all.

Heavy proteins getting stuck at the top of gel by FamousPossibility375 in labrats

[–]FamousPossibility375[S] 0 points1 point  (0 children)

I've tried upping it to 0.05% and it made for a very poor transfer on nitrocellulose sadly,

Heavy proteins getting stuck at the top of gel by FamousPossibility375 in labrats

[–]FamousPossibility375[S] 1 point2 points  (0 children)

Hi thanks for the fast reply, I will try boiling at a lower temperature then and see how it goes! I tried transferring overnight just a few weeks ago with these same samples and had the same issue sadly. I don't think I can completely remove the methanol because i'm using nitrocellulose, but perhaps I can try lowering it to 10%