Capillary sodium dodecyl sulfate-DALT electrophoresis of proteins in a single human cancer cell

Abstract

Capillary Sodium dodlecyl sulfate (SDS)-DALT an (abbreviation for Dalton) electrophoresis was applied to analysis of proteins in single HT29 human colon adenocarcinoma cells. A vacuum pulse was employed to introduce a single cell into the coated capillary. Once the cell was lysed, proteins were denatured with SDS, fluorescantly labeled with 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ), and then separated by using 8% pullulan as the sieving matrix. This method offers a few advantages for single-cell protein analysis. First, it provides reproducible separation of single-cell proteins according to their size. Based on comparison with the migration time of standard proteins, most components from a single HT29 cancer cell have molecular masses within the range of 10-100 kDa. Second, as a one-dimensional separation method, it gives fairly good resolution for proteins. Typically, around 30 protein components of a single HT29 cell were resolved, indicating that this method has similar peak capacity to SDS-polyacrylamide gel electrophoresis (PAGE). Third, this method shows high detection sensitivity and wide dynamic range, which is important because of the wide range of protein expression in living systems. Detection limits for standard proteins ranged from 10(-10) to 10(-11) M. Finally, this method provides much higher speed than classical gel electrophoresis methods, and it provides automated anlysis of cellular proteins at the single-cell level; the separation is complete in 30 min and the entire analysis takes approximately 45 min.

Coaxial Flow System for Chemical Cytometry

“…[8][9][10][11][12][13][14][15][16][17][18] Unfortunately, one disadvantage of CE as a tool for chemical cytometry is its relatively low throughput, typically 5-35 cells per day. 10,11,17,19 In order to fully characterize many biological attributes, the analysis of hundreds to thousands of cells is often necessary to obtain statistically relevant data. For example, 100's of Xenopus oocytes were analyzed to reveal the on/off switch of MAP kinases and 1000's of rat basophilic leukemia cells were analyzed to deconvolute specific signaling mechanisms of protein kinase C. 20,21 In order to improve the low throughput of the CE analysis of adherent cells, and increase the feasibility of implementing this technique routinely in evaluating cellular attributes, new highthroughput methods are needed for chemical cytometry performed by CE.…”

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

“…In particular, we are interested in developing a technology to allow the study of protein expression in single somatic cells. Currently, LIF is the most common detector for CE analysis of proteins in single cells [13][14][15][16][17][18][19][20].…”

Polymer sieving matrices in microanalytical electrophoresis

“…Pullulan was demonstrated in CE-format SDS protein electrophoresis assays. 96,101 The low viscosity of pullulan was a chief selection criteria.…”

“…96 An 8% pullulan sieving matrix yielded similar separation performance to that obtained by slabgel SDS-PAGE. 45 Griebel et al reported a 2D gel capillary electrophoresis (2D-CGE) in a PMMA microuidic chip. 46 The rst dimension was isoelectric focusing (IEF) using an immobilized pH gradient (IPG).…”