Dear all,
I have been having problems with various antibodies in my western blots.
For some reason they remain on the blot even after they were washed off (0.5% TBST 3x 5 minutes). I have tried doing more washes (0.5% & 1% TBST) and use antibodies from different species. The amount of antibody I use is relatively low (1:2500) and I always block with BSA. The antibody is diluted in 5% BSA and the membrane is PVDF. The antibodies are incubated at 4 degrees overnight, while the secondary is incubated for one hour on a rocker at room temperature. For imaging i use chemiluminescence HRP substrate. As of late the bands even started showing on the blot itself without any probing of antibodies (imaged attached).
Any suggestions would be greatly welcomed.
Many thanks.
You should blot for your protein of interest first, than strip the membrane and blot for actin.
You can also try loading less extract per lane.
Dear Yoav,
Thank you for your suggestions. I have tried them both previously and was not successful.
Do you see the actin band when blotting for your POI, before adding the actin AB?
If yes than what you have is a non-specific band not an actin band.
No, I do not. I have blotted my POI prior to beta actin and was still able to see the band of the POI present when i tested for beta actin. The order of use does not seem to matter as any antibody I use remains on the blot. Membranes that had 3 different antibodies ( different origin species) used on them, presented at the end with 3 bands, one of each of the previous antibodies used.
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