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antibodies of interest coming form the same host troubleshooting

antibodies of interest coming form the same host troubleshooting - Western Blot Forum

antibodies of interest coming form the same host troubleshooting - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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Old 07-10-2013, 02:37 PM
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Question antibodies of interest coming form the same host troubleshooting



Hi all!

I have a question and I really hope someone could help:

I had one blot and wanted to check the expression of two different proteins on the same blot (mol weights are far from each other so no problem with that). So, I incubated my blot first with the first antibody of interest (let's call it "A") and after I developed the image, I incubated the blot O/N with the other antibody of interest ("B"). The thing is that both antibodies of interest come from the same host (rabbit), so, of course, my secondary antibody in both cases is anti-rabbit as well (the same). So, when I developed the image for the "B", I just saw the image of the first antibody ("A"). I understand this could be a possible outcome, although i had washed the blot after the "A" antibody really well.
Any ideas to avoid that problem?? (Stripping could help but unfortunately I cannot risk losing any protein) Do i just need to use different blots, one for each protein? :/

thanks!
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