I am working on photosynthesis mechanisms on a new alga strain that has no previous studies and want to separate a special LHC under a specific light enviornment. At first, I want to compare the protein on the thylakoid membrane of this strain with Chlamydomonas reinhardtii by SDS-PAGE, therefore to determine the specific protein of each band. However, I found this is not accurate unless I undertake immunoblotting steps further. If I use BN-PAGE to separate complex first and to compare the result with C. reinhardtii, is it possible for me to determine these complexes on the thylakoid membrane and find the special LHC by following 2D-PAGE electrophoresis?
Electrophoresis results are at best semi-quantitative. Starting with loading errors and gel-to-gel variability, there are many things that can go wrong. As with all dye-binding assays, colour yield depends on protein. If you have standards (in the same gel!), you'd at least get an idea what the error margins are.
If the staining is not too strong (in the linear range of the densitometer), you could scan the gels and integrate the peaks. Laser-based densitometers are better at this than simple scanners, because of a larger linear range (up to 4 AU). Even better results may come from fluorescent dyes like RuBPS and fluorescent scanners, simply because fluorescence starts from zero, whilst in absorbance you measure the small difference of two large numbers.
You could also cut out the bands, elute the bound dye and determine that photometrically.
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