Effect of Triton X-100 and Dtt Concentrations on Wide Range Two-Dimensional Gel Electrophoresis of Tissue, Cell and Fluid Proteomes

AbstractHigh-resolution separation by two-dimensional gel electrophoresis (2- DE) is still challenging due to the intrinsic behavior of proteins, principally throughout isoelectric focusing separation. It is often observed low resolution of proteins in the alkaline pH region when using wide range pH gradients. Herein, we show the effect of different concentrations of Triton X‑100 and DTT in the sample buffer on wide range pH (3-10) 2-DE profiles of three different biological samples as Trypanosoma cruzi cells, honey bee brain tissue and human saliva fluid. Higher resolution, number and intensity of spots were achieved when 85 mM DTT and 2.5% Triton X‑100 were employed for cell and tissue samples. No improvement was observed for fluid proteins, probably because water-soluble proteins do not require special conditions for extraction and prevention of precipitation during isoelectric focusing.

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Factors affecting the range of pH gradients in the isoelectric focusing dimennsion of two-dimensional gel electrophoresis: The effects of reservoir electrolytes and loading procedures

Electrophoresis ◽

10.1002/elps.1150060707 ◽

1985 ◽

Vol 6 (7) ◽

pp. 339-348 ◽

Cited By ~ 10

Author(s):

John A. A. Chambers ◽

Francesco Degli Innocenti ◽

Katharina Hinkelammert ◽

Vincenzo E. A. Russo

Keyword(s):

Isoelectric Focusing ◽

Gel Electrophoresis ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Ph Gradients ◽

Factors Affecting

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Reproducibility and quality assurance of two-dimensional gel electrophoresis of serum specimens.

Clinical Chemistry ◽

10.1093/clinchem/28.4.908 ◽

1982 ◽

Vol 28 (4) ◽

pp. 908-914 ◽

Cited By ~ 31

Author(s):

R P Tracy ◽

R M Currie ◽

D S Young

Keyword(s):

Quality Assurance ◽

Gel Electrophoresis ◽

Sodium Dodecyl ◽

Two Dimensional ◽

Protein Loss ◽

Serum Samples ◽

Silver Stain ◽

Two Dimensional Gel Electrophoresis ◽

Report Quality ◽

Wide Range

Abstract Currently we are using two different ISO-DALT two-dimensional gel electrophoresis systems, designated MC-Iso 1 and MC-Iso 2, for the analysis of serum and plasma samples. Here we report quality-assurance data for both of these systems. CV values for the slopes of the pH gradient (ISO dimension) are 5.6% of less; CV values for the slopes of the molecular-mass curves (log Mr vs relative mobility in the DALT dimension) are 3.4% or less. We examined the various steps of the analysis in detail for reproducibility and protein loss, using radiolabeled albumin, alpha 2-macroglobulin, and beta 2-microglobulin. Generally, in the first dimension, less protein enters the MC-Iso 2 gels (our routine system in which silver stain is used) than enters the MC-Iso 1 gels (our wide-range system for myeloma serum samples, in which the gel is stained with Coomassie Blue), on the average, 87% as much. The CV at this stage for both systems is 5--8%. During equilibration, considerable amounts of protein are lost (approximately 30% in 10 min) from the ISO gel, and the reproducibility is also decreased. Resolution in the DALT dimension has, in most cases, little or no effect on either recovery or reproducibility. Overall, for most proteins expected to appear in an ISO gel of a given pH range, approximately 50--60% of the starting material may be expected to reside in the sodium dodecyl sulfate slab gel, under our conditions. The two most important variables affecting recovery are the concentration of the NaOH (used as catholyte) and the pH of the starting sample. The overall CV for the process is between 8 and 12%.

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