I will be running western blotting using NR2A and NR2B polyclonal antibodies for my proteins of interest, and I accidentally bought a monoclonal beta-tubulin antibody that I will use for a loading control. Antibodies can be expensive, and my grant is limited, so would it be kosher to proceed with analysis using a monoclonal antibody loading control versus polyclonal NR antibodies? Wouldn't this just reduce the signal of the loading control relative to the proteins of interest?
Hi Caroline,
It shouldn't be a problem to use antibodies of different clonality in this case. You just want to compare de loading of protein among samples/conditions. The signal you'll get could be reduced or increased but this will happens for all the samples under same antibody conditions, so the loading control remains a measure of the relative amount of protein loaded for each condition.
Cheers!
No, it likely won't be approved by a rabbi in time. Lucky for you, clonality won't be a problem for your western blot; you're just measuring the relative luminescence per lane, and there is so much beta-3-tubulin in the brain that you will likely need to dilute your primary antibody in order to see nice bands. Additionally, you can also normalize to the total protein loaded (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567873/).
Ha! I appreciate the joke, John. Thank you both for your valuable answers.
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