Bio-rad Laboratories, Inc.

Steve Freeby

Aran Paulus

Ning Liu

Anton Posch

Kevin McDonald

John Walker, Ii

Katrina Academia

Tim Wehr

John A Walker Ii

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.

Stain-free protein quantification and normalization

Phosphorylated peptides are extracted from digests of biological liquids and other peptide mixtures by fractionation on ceramic hydroxyapatite. The ceramic hydroxyapatite is readily usable in a centrifuge, allowing for rapid fractionations of a large number of small volume samples, and accordingly high throughput.

Phosphopeptide enrichment of compositions by fractionation on ceramic hydroxyapatite

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Vacaville, CA, United States of America

Steve Freeby