Disclosed herein are methods of
protein quantification and normalization using haloalkylated
tryptophan fluorescence.
Complex protein samples, i.e., samples that each contain 1,000 or more distinct proteins, from diverse sources that do not have common
protein profiles are treated with a halo-substituted
organic compound (i.e.
haloalkane) that reacts with
tryptophan residues to form fluorescent products.
Irradiation of the samples with
ultraviolet light and the detection and quantification of the
resultant fluorescent emissions from all proteins in each sample are then used to obtain comparative values for
total protein content among the various samples. The values thus obtained are found to be valid indications of comparative
total protein content, despite the fact that the
tryptophan levels vary widely among the various proteins in any
single sample and the samples, due to the diversity of their origins, tend to differ among themselves in the identities and relative amounts of the proteins that they contain.
Protein samples are also normalized to correct for differences in
sample dilution, sample loading, and
protein transfer inconsistencies, by using
stain-free detection of
total protein in each of the samples, or detection of subsamples within each sample.