I used strong RIPA to make samples from mouse liver, then adjusted protein concentrations by BCA. Finally I used 2x loading buffer and boiled samples.
I runned 12%, 13.5%, 15% gels, 90v -> 120v. but neither I cant get clear LC3B bands. In attached files, I run 13.5%gel but LC3B-I and II seemed very strange. Transfering condition is 300mA 60min. 5% nonfat milk to block.
The primary antibody used is Novus LC3B antibody in 1: 1000, which worked well on my colleague Hela cell samples. Anti rabbit is used in 1:10000.
It may have been a transfer problem...?
Could you do an overnight transfer with lower voltage in a cold room?
Best of luck!
Glycosylation of some proteins results in bands that are "fuzzy" in western bots. Is your target glycosylated?
I think it's liver to blame - not your conditions. I'm surprised you've got it that well with such a tricky sample source. Liver is notoriously fatty (granted, there are worse culprits, like fat tissue) and that can be a problem for short polypeptides like LC3B. I don't believe it's your transfer or glycosylation to that matter. The only thing that comes to mind is further dilution of your sample with 1X sample buffer and loading less - you can easily test it with one of your samples and a quick western. Do few sequential dilutions. It's a double-edge sword, however, as you might lose your signal faster than sharpen it. But I would say it's a fat that is a culprit. Those aren't HFD or ob/ob mice by any chance? Old age could do it too. Anyway - I'd say a good job, granted the circumstances. You do come across as a perfectionist however - a tough but appreciated quality in science. Good luck!
Shijie Fan I am curious which transfer membrane you used? Use 0.2 µ PVDF membrane instead of using 0.45 µ Nitrocellulose membrane.
Craig Silver
Thanks for answer!
I think this is not because of glycosylation. Other reseacher can get pretty LC3B bands.
Mudassar N Mughal
Thanks!
I used RIPA containing SDS so I think all protein went through denaturation.
Vadim Markovtsov
Thanks for your detailed answer! Actually these mice had problems in liver, you are really professional!
I will try to low sampling concentration to get better result.
Shijie Fan 0.2 µ pore size is very important.
If liver fat is the issue during protein sample preparation from liver tissue you can fractionate out the fat and only work with the protein in it. We often use this technique when we perform western blot with Drosophila fat body tissues (which is liver equivalent to mammal).
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