Hello,
I ran 50ug whole cell extracts of HEK293 on Western and probed for yH2AX mouse overnight 4C (Image 1). After developing I probed the same blot with tubulin mouse again overnight 4C (Image 2). I develop my blots with 1X Luminol solution activated with H2O2.
Because the antibodies are from the same species, I see yH2AX signal showing up when developing the blot second time. My question is why is that the pattern of yH2AX signal different?
All help is appreciated.
Thanks
My opinion is that plots developed with the HRP reaction should not be re-probed because of oxidative damage by the formation of peroxide. It would be better to make two identical blots, one for each antibody, or use both antibodies at the same time to probe one blot.
I agree with Adam B Shapiro.
You will have at methionine oxidized and cysteine residues oxidized, even disulfide cysteines pare further oxidized to sulfonic acid or even persulfide. Other residues could be attacked, too.
Hi Bakhtiyar Taghizada . Are the MW markers in the far left lane also showing up positive?
Hi Steingrimur Stefansson,
My MW marker does not show up positive when imaging chemiluminescence. It is from a colorimetric image that I then merged with chemiluminescent image.
Thanks for your responses. I would understand if some of the signals are lost due to imaging second time, however why is that in my first lane (lane right to MW) the signal is actually much more visible in Image 2? Image 1 is 60 sec exposure and Image 2 is 100 sec exposure. However, the first lane in the Image 1 showed only faint signal even after exposing 600 sec.