I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software.

My questions are about what estimates can be made, given that Coomassie staining can be nonlinear has protein-to-protein variability. Unfortunately, I do not have a standard curve, and the lanes contain total lysate.

  1. Can I take the percentage of band volume to lane volume as an estimate of percentage of total cell protein mass? (25% vs 20% total cell protein)

  2. Or would it more appropriate to take the band volume normalized to an arbitrary band in the lysate, and then report the percentage difference between conditions? (6 vs 4.8 normalized band volume, or 20% decrease in expression levels).

  • 1
    $\begingroup$ I don't think you can get away with either. You have no way of knowing that a specific band represents a single protein. Sounds like you should be doing a western blot. $\endgroup$ – canadianer Nov 1 '15 at 22:23
  • $\begingroup$ @canadianer Even western blots have nontrivial levels of background staining (especially for polyclonal antibodies), so one will still need to run a standard curve. $\endgroup$ – March Ho Nov 1 '15 at 22:41
  • $\begingroup$ @MarchHo that depends on the antibody... $\endgroup$ – MattDMo Nov 2 '15 at 0:38

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Browse other questions tagged or ask your own question.