The experimental condition I used: HUVECs, (p5-p6). I transfected them with either control or mimic and collected protein in RIPA. I loaded 20ug protein on each well, I tried with 10%, 12% and 15% gel, either transfer overnight at the cold room with 25V, or transferred on ice 100V for 1hr. I usually use 3% BSA for blocking, 1hr at room temperature and incubate with primary antibody (anti-p62 from cell signalling) overnight in the cold room. I consistently failed to obtain p62 bands both in the control and treatment groups. Other protein worked fine. It would be greatly appreciated if anyone can provide some suggestions, thanks!
The protocol sounds well planned/designed.
In my opinion, the antibody is possibly not specific (well tested/suited) for your protein of interest (for ref. see Baker M. Reproducibility crisis Blame it on the antibodies. Nature. 2015_521.p274-6. doi 10.1038521274a). If you have the possibility to test the antibody (e.g. via utilizing recombinant p62 protein) you could test this idea.
Another possible source of uncertainty is also the experimentator self.
Dear Shuhan Bu
I havent work with HUVEC cells but in my previous work, I used MCF-7, MDA-MB-231 and BT-474 cells and detected p62. We used cst #5114 p62 antibody and it worked really well. Our gel concentration was 12% but I dont think it will affect your detection. Overnight transfer at the cold room with 16V for 16h or transfer on ice with 100V for 120 min also worked. We blocked the membrane with 5% non-fat dry milk in TBS-T and our antibody concentration was 1:500 but 1:1000 also worked.
If you need more detailed protocol, here is our latest publication: https://www.mdpi.com/1420-3049/26/4/854/htm
Good luck
Hi,
this sound like antibody is your problem. I faced this problem with a lot of antibodies. Try diluting you antibody in TBS-BSA-NaN3, TBST-BSA (3%), or 5% non-fat milk in TBST and test in on WB. I found that some antibodies require specific diluting solution.
Hi,
It could be possible that your antibody is not working well. I used anti-P62 from abcam (ab56416) and it always worked fine in BV2 microglial cells. I followed the following protocol:
Use 12% gel, load 30ug protein in each well, transfer on ice 100V for 1hr, use 5% milk PBST for blocking 1.5 hours at room temperature (or overnight in cold room), dilute the primary antibody in 1% milk PBST and incubate for 1.5 hours at room temperature with gentle shaking (incubation overnight in cold room also works well).
Good luck!
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