# US 10,119,961 B2 · Stain-free protein quantification and normalization > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,961 B2 - Document ID: US10119961B2 - Status: Granted - Kind: B2 - Application number: 15/293,154 - Filed: 2016-10-13 - Published: 2017-02-02 - Granted: 2018-11-06 - Earliest claimed: 2012-04-27 - Assignees: Bio-Rad Laboratories, Inc. - Applicants: Bio-Rad Laboratories, Inc. - Inventors: Steve Freeby; Ning Liu; Kevin McDonald; Aran Paulus; Anton Posch - Web page: https://patentgrep.com/patent/US10119961B2 ## CPC - G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation - G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor - G01N 33/6827: {Total protein determination, e.g. albumin in urine} - G06T 7/0012: {Biomedical image inspection} - G06T 11/60: Creating or editing images; Combining images with text - G16B 99/00: Subject matter not provided for in other groups of this subclass - G06T 2207/30004: Biomedical image processing - G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL ## Abstract 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. ## Claims ### Claim 1 A method of normalizing, using detected haloalkylated tryptophan fluorescence, the amount of protein in a first subsample of a protein sample to the amount of protein in a second subsample of the protein sample, the method comprising: quantifying the amount of haloalkylated tryptophan fluorescence detected from the first subsample; quantifying the amount of haloalkylated tryptophan fluorescence detected from the second subsample, wherein the second subsample comprises 50 or more proteins; and normalizing the amount of protein in the first subsample to the amount of protein in the second subsample based on the amounts of haloalkylated tryptophan fluorescence detected from the first subsample and the second subsample. ### Claim 2 (depends on 1) The method of claim 1, wherein the first subsample is one protein. ### Claim 3 (depends on 1) The method of claim 1, wherein the second subsample is all proteins in the sample. ### Claim 4 (depends on 1) The method of claim 1, wherein said normalizing is performed on a computer. ### Claim 5 (depends on 1) The method of claim 1, wherein said normalizing comprises dividing the amount of haloalkylated tryptophan detected from the first subsample by the amount of haloalkylated tryptophan detected from the second subsample to obtain a ratio. ### Claim 6 (depends on 5) A method of comparing the relative amounts of a target protein in a first protein sample and a second protein sample, the method comprising normalizing, according to claim 5, the amount of the target protein in the first protein sample to the amount of all proteins in the first protein sample, to obtain a ratio for the first protein sample; normalizing, according to claim 5, the amount of the target protein in the second protein sample to the amount of all proteins in the second protein sample, to obtain a ratio for the second protein sample; and comparing the ratios for the first and second protein samples, wherein all proteins in the first protein sample are 50 or more proteins and all proteins in the second protein sample are 50 or more proteins. ### Claim 7 (depends on 1) The method of claim 1, further comprising the step of obtaining an image of an electrophoresis gel or blotting membrane comprising the protein sample, and wherein the amount of haloalkylated tryptophan fluorescence detected from each subsample is quantified using the image. ### Claim 8 (depends on 7) The method of claim 7, wherein the image is obtained by contacting the protein sample with a haloalkane, exposing the electrophoresis gel or blotting membrane to UV light, and recording fluorescently re-emitted light having a wavelength of from about 400 nm to about 500 nm. ### Claim 9 (depends on 7) The method of claim 7, wherein the amount of haloalkylated tryptophan fluorescence detected from each subsample is quantified by: designating a portion of the image to correspond to the subsample; and determining the total amount of haloalkylated tryptophan fluorescence arising from the subsample in the portion of the image. ### Claim 10 (depends on 9) The method of claim 9, wherein the total amount of haloalkylated tryptophan fluorescence arising from each subsample is determined by integrating the haloalkylated tryptophan fluorescence in the corresponding portion of the image over the area of that portion of the image. ### Claim 11 (depends on 9) The method of claim 9, further comprising performing a background subtraction step comprising: from the electrophoresis gel or blotting membrane comprising the protein sample, obtaining a background image, wherein the background image corresponds to a part of the gel or membrane that contains substantially no protein; determining the total or average amount of haloalkylated tryptophan fluorescence in the background image; and subtracting the total or average amount of haloalkylated tryptophan fluorescence in the background image from the total amount of haloalkylated tryptophan fluorescence in each portion of the image.