I'm interested if a protein is being degraded. My idea is to western blot my samples, add an antibody for ubiquitin, image, wash off the antibodies, add an antibody for the protein of interest, image, then subtract the images from each other to show only the ubiquitination of the protein of interest.
My concern is that due to survivorship bias, this will not show what I want it to show. I want to know if the protein is being degraded, but what this would show in reality is the proteins that have been ubiquitinated, but have not been degraded. Should I be concerned about this? Are there any better methods for this purpose?
To ensure your protein is ubiquitinated but not degraded in this scenario, I advise using MG132 (proteasomal inhibitor) in your experimental conditions according to a required dose. Then perform a western blot and ubiquitin antibody check. So you can get only ubiquitinated proteins. I think this will solve your question.
Best of Luck
As stated above MG132 is a very good proposal. In additon, you could perform immoprecipitation for your protein of interest and in the resulted samples, Wwstern blotting against ubiquitin.
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