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2-D Electrophoresis

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Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.

This technique separates proteins according to two independent properties in two discrete steps:

  • The first dimension step, isoelectric focusing (IEF), separates proteins according to their isoelectric points (pI).
  • The second dimension step, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), separates proteins according to their molecular weights (MW).

Each spot on the resulting two-dimensional array corresponds to a single protein in the sample. Thousands of different proteins can be separated, and information such as the pI, apparent MW, and the amount of each protein can be obtained.

Applications General Considerations

2-D Electrophoresis protein spot map.

Proteomics—the systematic separation, identification, and quantitation of many proteins simultaneously from a single sample—relies on 2-D electrophoresis and its exceptional ability to separate thousands of proteins at once. 2-D electrophoresis can also be used to detect post- and co-translational modifications, which cannot be predicted from the genomic sequence.

Other applications of 2-D electrophoresis include analysis of cell differentiation, detection of disease markers in research, drug discovery research, cancer research, purity checks, and microscale protein purification.

2-D Electrophoresis protein spot map.

Sample Preparation
Appropriate sample preparation is absolutely essential for good 2-D electrophoresis results. The samples must have the right composition for IEF, which means they must be in a solution that does not affect the pI of the proteins to be separated. In order to get optimal results, the samples should not include impurities, especially ionic impurities. Sample preparation can be improved by using specific products that both clean-up the samples and leave them in optimal buffers for gel electrophoresis.

One potential problem with 2-D electrophoresis is streaking. This is due to nonspecific oxidation of thiol groups on proteins. Using specific reagents to eliminate this will reduce streaking between spots in the gel, especially in the pH range 7 to 11, and will also simplify the spot pattern by reducing the number of spots caused by protein oxidation.

Spot Processing
Gel spots of interest can be picked and analyzed by mass spectrometry (MS) to identify the corresponding proteins. The procedure of picking and digesting spots can be performed manually or semi-automatically by manual transfer of gels and microplates between instruments, or automatically using an integrated workstation.

Ettan Spot Picker is designed to reproducibly spot protein samples and matrix on multiple MALDI MS targets for subsequent analysis by mass spectrometry.

Ettan Digester is a highly versatile instrument designed to perform in-gel digestion of proteins captured in 2-D gel electrophoresis spots.

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