Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells

Abstract

Capillary sodium dodecyl sulfate (SDS)-DALT electrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, separated by SDS-DALT-CE in polyethylene oxide sieving matrix, and detected by laser-induced fluorescence (LIF) in a sheath flow cuvette. This method was combined with detergent differential fractionation, which is a protein fractionation method using a series of detergent-containing buffers to sequentially extract protein fractions from cells, to analyze the proteins in HT29 human colon adenocarcinoma cells. In addition, on-column labeling was demonstrated for protein analysis by SDS-DALT-CE with LIF, and applied to analysis of proteins in a single HT29 cancer cell. Most proteins had molecular masses from 10 to 120 kDa. Similar protein profiles were obtained for single cells and protein extract of a large cell population.

CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals

“…Orange G is a negatively charged marker commonly used in capillary gel electrophoresis applications for determination of protein molecular weight (MW). It has a small MW and migrates faster than protein-SDS molecules through sieving matrices [18]. In this study, OG was chosen in order to simulate the behavior of the negatively charged protein-SDS molecules.…”

“…The apparent net charge carried out by a protein molecule depends on the isoelectric point (pI) of the protein and pH of the BGE employed [11]. The use of SDS in conjunction with a reducing agent is a commonly used procedure for preparation of protein samples for both conventional and capillary gel electrophoresis [18,19]. This pre-treatment effectively imparts a negative charge on protein molecules proportional to the mass of the protein (&1.4 g SDS/g protein).…”

“…This pre-treatment effectively imparts a negative charge on protein molecules proportional to the mass of the protein (&1.4 g SDS/g protein). Protein-SDS complexes migrate at the same speed under the effect of an applied electric field [18][19][20][21].…”

Capillary Electrophoresis for the Analysis of Biopolymers

“…Our group reported a series of papers applying CSE methods for highly sensitive protein analysis for cell homogenates and single cells using 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic reagent (31)(32)(33). The labeled proteins were separated in a replaceable polymer sieving matrix such as PEO or pullulan and then detected using an ultrasensitive sheath-flow laser-induced fluorescence detector.…”

In-Gel Staining of Proteins in Native Poly Acryl Amide Gel Electrophoresis Using Tetrakis(4-sulfonato phenyl)porphyrin

“…Further complicating attempts to label proteins by fluorescent tags is the requirement that both protein and dye are present in higher concentrations for the reaction to proceed, 4 although exceptions have been reported. 5 Non-covalent fluorescent labelling and quenching of proteins has been an important method in the study of protein folding. 6 Non-covalent labels used thus far include indocyanine green, 7 squarylium dyes, 8 NanoOrange, 9 and the SYPRO series of dyes.…”