Prefractionation of protein samples prior to two‐dimensional electrophoresis

Thousands of proteins may be visualised on a two‐dimensional (2‐D) gel, but only hundreds are present at levels sufficient for chemical analysis. Therefore, prefractionation of protein samples prior to 2‐D polyacrylamide gel electrophoresis (PAGE) will be important for the investigation of proteins that are present at sub‐picogram levels in physiological samples. We describe an approach to prefractionate protein samples prior to 2‐D PAGE using the Gradiflow, which is a new (preparative) electrokinetic membrane apparatus designed to fractionate proteins in a number of different ways. We have fractionated human serum under nonreducing conditions using the ‘reflux’ mode, in which proteins are fractionated according to their relative mobility under controlled electrophoretic conditions, where the current is periodically reversed. We describe how fractionation occurs and present examples of enrichment of specific proteins.

[1]  J. Kyhse-Andersen.  Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. , 1984, Journal of biochemical and biophysical methods.

[2]  Ettore Appella,et al.  Methods in Protein Structure Analysis , 1995, Springer US.

[3]  M R Wilkins,et al.  Large-scale amino-acid analysis for proteome studies. , 1996, Journal of chromatography. A.

[4]  Thierry Rabilloud,et al.  Sample application by in‐gel rehydration improves the resolution of two‐dimensional electrophoresis with immobilized pH gradients in the first dimension , 1994, Electrophoresis.

[5]  B. Honoré,et al.  Bladder squamous cell carcinomas express psoriasin and externalize it to the urine. , 1996, The Journal of urology.

[6]  D. Hochstrasser,et al.  Micropreparative two‐dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins , 1993, Electrophoresis.

[7]  L. Hood,et al.  Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis. , 1986, The Journal of biological chemistry.

[8]  J I Garrels,et al.  Quantitative exploration of the REF52 protein database: Cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation , 1990, Electrophoresis.

[9]  G. Corthals,et al.  Multifunctional apparatus for electrokinetic processing of proteins , 1994, Electrophoresis.

[10]  R. Aebersold,et al.  Mass spectrometric approaches for the identification of gel‐separated proteins , 1995, Electrophoresis.

[11]  T. Rabilloud.  A comparison between low background silver diammine and silver nitrate protein stains , 1992, Electrophoresis.

[12]  Joachim Klose,et al.  Two‐dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome , 1995, Electrophoresis.

[13]  M R Wilkins,et al.  Rapid protein identification using N-terminal "sequence tag" and amino acid analysis. , 1996, Biochemical and biophysical research communications.

[14]  P. Matsudaira,et al.  Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. , 1987, The Journal of biological chemistry.

[15]  Complexity of sea urchin embryo nuclear proteins that contain basic domains. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  B. Wittmann-Liebold.  Methods in Protein Sequence Analysis , 1989, Springer Berlin Heidelberg.

[17]  J. Vandekerckhove,et al.  Protein-Electroblotting and Microsequencing in Establishing Integrated Human Protein Databases , 1990 .

[18]  Morten Østergaard,et al.  The human keratinocyte two‐dimensional gel protein database (update 1995): Mapping components of signal transduction pathways , 1995, Electrophoresis.

[19]  R. Aebersold,et al.  Characterization of Proteins Separated by Gel Electrophoresis at the Primary Structure Level , 1995 .

[20]  D. Hochstrasser,et al.  Improved and simplified in‐gel sample application using reswelling of dry immobilized pH gradients , 1997, Electrophoresis.

[21]  G. Corthals,et al.  Preparative reflux electrophoresis , 1995, Electrophoresis.

[22]  Angelika Görg,et al.  Two‐dimensional electrophoresis. The current state of two‐dimensional electrophoresis with immobilized pH gradients , 1988 .

[23]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[24]  D. Hochstrasser,et al.  Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. , 1996, Biotechnology & genetic engineering reviews.

[25]  D. Hochstrasser,et al.  From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Arnino Acid Analysis , 1996, Bio/Technology.

[26]  D. Hochstrasser,et al.  A nonlinear wide‐range immobilized pH gradient for two‐dimensional electrophoresis and its definition in a relevant pH scale , 1993, Electrophoresis.

[27]  G. Corthals,et al.  The role of pH and membrane porosity in preparative electrophoresis , 1996, Electrophoresis.

[28]  D. Hochstrasser,et al.  Characterization of Human Plasma Glycoproteins Separated by Two-Dimensional Gel Electrophoresis , 1996, Bio/Technology.

[29]  J. Celis,et al.  A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype , 1994, Electrophoresis.