Is analyzing soluble protein content in pollen grains using the Bradford assay and visible spectroscopy feasible? By preparing the pollen extract in phosphate buffer (pH 7.4) and mixing it with the Bradford reagent, a measurable color change at 595 nm occurs, effectively quantifying the protein content in pollen.
It should be feasible to use the Bradford assay to measure the soluble protein concentration of a pollen extract after removal of insoluble material. You require a standard protein to make a standard curve. Ideally, the standard would have an amino acid composition similar to that of the extract in order to be accurate. In the absence of such a standard, you can still get a useful relative measurement using bovine serum albumin as the standard.
Do a Bradford assay as Adam mentioned, after clearing your extract by centrifugation or filtration. But for the love of god, do the A595/A465 ratio method. It has been around for over 30 years and people still do sole A595, probably right after waddling to the lab in Fred Flintstones car.
Article Linearization of the Bradford Protein Assay
It got to be said, quantifying proteins is always a rule of thumb thing. The only true way of quantifying a protein is gravimetry, which is near impossible on an analytical scale. Depending on your protein composition (as Adam mentioned) you may end up with measured concentrations that may be off by a factor of 2 or seldomly even more. No matter whether you do A280, Bradford, BCA or any other measurement, proteins are hardly standardized.
Andre Marschall Thank you for referring to this article on ratiometric measurement in the Bradford assay. I was not aware of it before.
Using commercially prepared Bradford reagent (either from Bio-Rad or Amresco), I've found the micro version of the assay (10 µl sample + 200 µl dye in a 96-well microplate) to be linear up to 5.5 µg of BSA using just the 595 nm measurement, and second-order up to 11 µg.
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