Protein Sample Preparation for 2-D Electrophoresis

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Overview

Efficient and reproducible sample preparation methods are key to successful 2-D electrophoresis. Sample preparation methods range from extraction with simple solubilization solutions to the use of complex mixtures of chaotropic agents, detergents, and reducing agents. Sample preparation can include enrichment strategies or separating protein mixtures into reproducible fractions to reduce their complexity.

Related Topics: Protein Extraction and Cleanup, Protein Fractionation and Depletion, 2-D Electrophoresis, and Troubleshooting 2-D Gels with 2-D Electrophoresis Doctor™.

 

Solubilization

An effective sample preparation procedure will:

  • Completely lyse the cells or tissues to release the protein content
  • Reproducibly solubilize proteins of all classes, including hydrophobic proteins
  • Prevent protein aggregation and loss of solubility during focusing
  • Prevent post-extraction chemical modification, including enzymatic or chemical degradation of the protein sample
  • Remove or thoroughly digest nucleic acids and other interfering molecules
  • Yield proteins of interest at detectable levels, which may require the removal of interfering abundant proteins or non relevant classes of proteins

 

Most protein extraction projects will require empirical determination of optimal conditions for your 2-D PAGE application. Variations in the concentrations of chaotropic agents, detergents, ampholytes, and reducing agents can dramatically affect the 2-D pattern. Find sample preparation and protein quantitation methods in the Protocols section below.

Related Content

2-D Video Tutorial
How to run a 2-D Gel from start to finish.
 
Literature
Number Description Download
2651 2-D Electrophoresis for Proteomics: A Methods and Product Manual, Rev F Click to download
2895 Protein Blotting Guide, Interactive PDF, Rev B Click to download
Number Description Options
6200 Sample Quantitation (RC DC Protein Assay) Protocol and Compatibility Table Click to download
6221 Sample Quantitation (RC DC Protein Assay) Click to download
6220 Solubilization Click to download
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