Hello,
I incubate two type of antibodies A and B together in the same membrane. Both are same species monoclonal, but bind to different protein molecular weight. It usually give me the result in Western blot that I expect: A give a band signal at 60 kda and B is at 20 kda as mentioned in the antibody datasheet, so I never tried to incubate one antibody at a time.
However, one of my colleague incubate the membrane (protein from same type cell sample) with only antibody A (from same vial/tube) give one more band signal at 45 kda.
I tried to investigate what is the reason behind this additional band. Both I and my colleague did the experiments in the other steps with same protocol, except my colleague sample are very old protein lysate, store at -20 degree C for more than 6 month, and mine is a few day protein lysate, store at -80 degree C.
Is it because of probing multiple primary antibodies together that the 45 kda band signal isn't detected?
Thank you so much
Cheerio,
Your colleague's sample has probably broken down due to protease action and now gives two bands. Normal one at 60 kDa and break-down product at 45 kDa. Do not think it has anything to do with combined antibodies.
Using multiple primary antibodies simultaneously is done very commonly and should not prevent protein signal from being detected.
I think your colleague's old lysate has undergone proteolytic degradation and hence he is seeing two bands.
Proteins behave better when stored at -80 compared to -20 degrees.
- Badges
- Science method