Is it ok to probe with antibodies of different clonalities at the same time? For instance, if I'm using a mouse monoclonal GSK3 antibody and also want to use a rabbit anti mouse polyclonal GAPDH antibody, would anything be disrupted if they were run together? As long as I'm using appropriate secondaries (rabbit anti mouse for the monoclonal primary and goat anti rabbit for the polyclonal GAPDH primary) will signal be able to be detected?
Thank you all so much for your insight.
I've run both together, but my loading control (polyclonal) didn't show up very well. However, my loading control didn't show up very well in ANY of the westerns I ran, even when it was run along with another polyclonal antibody, so that was likely my problem.
Specifically, I ran a tau-5 (total tau) monoclonal antibody with a cytochrome C polyclonal antibody. My total tau bands looked beautiful, so at the very least nothing odd was happening there. I've never needed to try this with any other antibodies, so I'd be interested in hearing others experiences as well.
Have you already tried this without results, or are you planning on running it in the future? Look forward to seeing the results!
I also ran a monoclonal B-tubulin with polyclonal NR2A and NR2B. My NR2B antibody was solid, my NR2A antibody was "meh", and the monoclonal B-tubulin signal was extremely weak. So - same results as Kristen: good/okay probe of interest; not-so-great loading control.
Interesting that the loading control in each case is the one showing up "not-so-great". Maybe it's worth stripping and reprobing or running a running control in a separate gel but at the same time (i.e. dual chamber system)
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