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 μL assay displayed a sensitivity of 10.5 μg in a range up to at least 200 μg. Furthermore, we extended the utility of this method through the development of a 20 μL low-volume assay, with a sensitivity of 8.7 μg tested up to 100 μg, 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.

A rapid method for protein staining in polyacrylamide gels using water saturated with chloroform

“…The chloroform staining method is also a good alternative for trichloroethanol-based protocols. Even though the protocol requires to use proteins containing tryptophan, we believe that in time it will be commonly used for standard electrophoresis based procedures, such as protein characterization by means of 2D electrophoresis, analysis of protein transfer during western blotting and other laboratory procedures that require protein gel staining (Ladner et al, 2004;Gürtler et al, 2013;Chopra, Willmore & Biggar, 2019).…”

The Human UGT2B7 Nanodisc

“…The 2B7-to-MSP stoichiometry was obtained from the relative fluorescent intensities of the 2B7 and MSP protein bands in UVexposed, SDS-PAGE gels infused with TCE. TCE undergoes UV-induced reactions with Trp and Tyr residues (Casas-Finet et al, 1992;Ladner et al, 2006) that allow them to be distinguished based on their fluorescence emission spectra (Chopra et al, 2019). The TCE reactions are covalent, highly efficient (Casas-Finet et al, 1992), and have been used to quantitate proteins in SDS-PAGE gels based on their Trp composition and fluorescent intensity relative to standards (Casas-Finet et al, 1992;Chopra et al, 2019).…”

“…Finally, to remove aggregates and nanodisc stacks, the solution was passed through a size exclusion column (Superdex 200 Increase 10/300) equilibrated at ambient temperature in phosphate buffer. UGT2B7 was quantitated by comparing its fluorescence to that of purified standards (MSP1D1, SULT2A1, SULT1E1, Hexokinase, and GST4A) in TCE-treated SDS-PAGE gels (Chopra et al, 2019). The purity of the final UGT2B7 preparation was estimated, using SDS PAGE, at ;95%.…”

An Efficient and Cost-effective Purification Methodology for SaCas9 Nuclease

“…280 ) is usually used for keeping track of protein elution. Although such an optical setup is not included in the reported method, SaCas9 can be directly visualized in the TCE-stained SDS-PAGE gels within 30 minutes of chromatography 18–20 . The second limitation is the absence of a pressure monitor for prepacked columns.…”