Hello everybody, I am a new member here and recently joined not only because I love science but also because I have this question to which I cannot find an answer. I recently conducted a research study on quantification of protein powders (like the ones advertised to bodybuilders/athletes). I am a high school student who worked in a lab, so I began with Bradford, but upon a background reading of the protein solutions alone (no assay), the absorbance was too great on the plate reader to even warrant further attempts. I then moved onto many different methods, such as gel filtration, ammonium sulfate precipitation, and lipid extraction. Essentially, the other ingredients (flavorings, colorings, thickeners, etc.) would get in the way of the method.
Eventually, I decided to dilute the solutions to a very low concentration in attempts to effectively make the other ingredients so diluted that they wouldn't interfere in the readings. I put the solutions in a spectrophotometer and attempted to read the absorbance at 280 nm and 260 nm, then use the equation Concentration = 1.55*A280 - 0.76*A260, which, from my research, I know is a method to estimate concentration of unknown proteins (peak absorbance at 280nm due to aromatic aminos like Tyrosine and Tryptophan) with possible nucleic acid contamination (peak absorbance at 260nm). What I could not understand, or find for that matter, is why the ratios of 1.55 and 0.76 are used. I assume they are absorbance coefficients, maybe even the average absorbance coefficients at that wavelength for all proteins, but I can't find an answer wherever I look.
SHORTER VERSION: In the equation for protein quantification, Concentration = 1.55*A280 - 0.76*A260, where do the coefficients 1.55 and 0.76 come from?
Thank you all for the help!
