I would like to compare the transgenic protein expressions in leaf and stem tissues of my transgenic Arabidopsis plant on Western blot. In the past, I've compared protein expression of the same tissue type by loading equivalent protein amount using RuBisCo LSU to normalize. Now I need to compare proteins in different tissue types, so I would like to know if it is still fair to using RuBisCo LSU to normalize and compare protein of different tissue types? Or should I normalize solely with Bradford? The EFP Browser showed that RuBisCo LSU (ATCG00490) is expressed differently in leaf (>1000) and stem (~600). Thanks in advance!
Rubisco should definitely not be used for normalising against different tissue types. Rubisco is only found in photosynthetically active cells containing the Calvin cycle. Your best bet would be to load equivalent amounts of total protein based on Bradford.
To identify a protein that you could use in Western blots to normalise concentration across different tissue types, you could run a gel containing equal amounts of protein from each tissue type, stain it and look for bands that are consistently of the same intensity across all tissue types. You could then excise the band and sequence it by mass spectrometer. This would help you identify the protein. Of course you would then hope that a commercially available antibody is available for the protein you identify.
Thanks! I was suspecting this approach because of that reason. Thanks for the confirmation! My transgenic protein has an HA tag, so I'm good on detection.
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