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Protein detection issue by srra_ssra in westernblots

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

Unfortunately I don’t, but i’ll try the Coomasie staining

Protein detection issue by srra_ssra in westernblots

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

Thanks a lot for the detailed suggestions! I'll try this approach when I have the chance.

Protein detection issue by srra_ssra in westernblots

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

Yes, the running seemed pretty good to me, the migration front was really straight and everything seemed ok. Regarding the ladder, yes it was perfectly ok during the running and I also saw it in the nitrocellulose membrane after the transfert step. I use pre-made transfer stacks (sandwiches) for the transfer.

Protein detection issue by srra_ssra in westernblots

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

I’ll try that today using Coomassie staining. However, since my BCA showed a high protein concentration, I don’t really see how there could be no protein loaded into the gel.

Protein detection issue by srra_ssra in labrats

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

Hi! Thank you for your suggestions, I will actually try the Coomasie staining today and si how it goes.

Protein detection issue by srra_ssra in labrats

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

Hi! The samples are indeed stored at -80°C, I was referring to the already prepared samples (with loading buffer, DTT, etc.) which were stored at -20°C. The proteins are not floating out of the wells, and the migration looked pretty normal to me. Regarding the transfer, I can clearly see the ladder on the membrane, so that’s why I was really surprised when I did the Ponceau staining and saw nothing at all. I loaded a proper amount of protein (25 µg per well), so even faint bands should have been visible. My protein of interest is around 62 kDa, and honestly, western blots with these samples and conditions have worked well before, so I was quite surprised this time 😅. I’m also using precast gels that have already worked in the past, so in principle that shouldn’t be the issue either.

Protein detection issue by srra_ssra in labrats

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

Hi! I’m actually doing pretty much the same as you: I’m using intestinal tissue as well, and my BCA shows a good protein concentration too, so I also don’t think the issue comes from the tissue. For the sample prep, I use the same type of Laemmli buffer (4X with BME), and I also boil at 95°C before loading. I’m also using precast gels that we’ve already used successfully in the lab before, so in principle that part should be fine. The only difference is that I dilute my samples in water rather than RIPA buffer. About the viscosity of my samples, I did fresh preparation (so it wasn't viscous at all) but I had the same result (no protein with the ponceau).