I used to normalize my target protein using housekeeping genes. But now I have come to know about total protein normalization. So I would like to do the same. I want to know how to measure total protein using Ponceau S stain.
@Saswat KUMAR Mohanty, after you transfer you proteins onto a membrane, stain the proteins bound to membrane with Ponceau S, then accurately wash the background to clearly visualise protein bands. Then scan the membrane stained with Ponceau (we use ChemiDoc from Bio-Rad), then estimate the total stain volumes per lanes (we do this in ImageLab software). After you finish the WB and get signals you can normalise the signals to the Ponceau S per exact lanes, where you observe your bands. Sometimes, depending on experiment, it gives more reliable values to compare
Saswat Kumar Mohanty once you've imaged your blot, you can use our Phoretix 1D software to represent bands as a % of total band volume in a lane (total protein visualised) and show the total volume of all proteins within each lane in the "results" section of the software. You can get a free trial of Phoretix 1D here - https://totallab.com/products/phoretix-1d/
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