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Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol.

ABSTRACT: The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal generated by modified proteins. We also demonstrated a specific fluorescent emission of 2,2,2-trichloroethanol-labeled protein at 450?nm, with a 310?nm excitation, resulting from modification of both tryptophan and tyrosine residues. Following optimization, this protein quantification assay displayed superior sensitivity when compared to UV absorbance at 280?nm (A280), and enabled quantification beyond the linear range permitted by the Bradford method. This 100??L assay displayed a sensitivity of 10.5??g in a range up to at least 200??g. Furthermore, we extended the utility of this method through the development of a 20??L low-volume assay, with a sensitivity of 8.7??g tested up to 100??g, which enabled visualization of proteins following SDS-PAGE. Collectively, these results demonstrate the utility of 2,2,2-trichloroethanol-based protein quantification and demonstrates the protein visualization in polyacrylamide gels based on 2,2,2-trichloroethanol-labeling pre-electrophoresis.

SUBMITTER: Chopra A

PROVIDER: S-EPMC6763483 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol.

Chopra Anand A Willmore William G WG Biggar Kyle K KK

Scientific reports 20190926 1

The incorporation of 2,2,2-trichloroethanol in polyacrylamide gels allows for fluorescent visualization of proteins following electrophoresis. Ultraviolet-light exposure, in the presence of this trichlorinated compound, results in a covalent modification of the tryptophan indole ring that shifts the fluorescent emission into the visible range. Based on this principle, we used 2,2,2-trichloroethanol to develop a microplate format protein quantification assay based on the fluorescent signal genera ...[more]

PMID: 31558752

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Dataset Information

Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol.

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Protein quantification and visualization via ultraviolet-dependent labeling with 2,2,2-trichloroethanol.

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