Analysis of Membrane Proteins by Two-dimensional Electrophoresis (2-DE) (CAT#: STEM-ET-0347-ZJF)

Two-dimensional gel electrophoresis (2DE) is a form of gel electrophoresis commonly used to separate and analyze proteins by molecular charge and molecular size. Proteins are first solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. In the first dimension, proteins are separated by their isoelectric point (pl) in isoelectric focusing (IEF). In the second dimension, proteins are separated by molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each protein is therefore resolved at a unique isoelectric point/molecular weight coordinate. After visualisation by staining proteome changes are revealed by gel image analysis, and protein spots of interest excised and identified by mass spectrometry sequence analysis combined with database comparison.

Molecular biology, proteomics, membrane proteins

1. Preparation: Proteins are usually solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. The procedures of sample preparation are dependent on the sample type.
2. Isoelectric focusing (IEF): In a particular pH gradient, proteins are separated by differences in their isoelectric points.
3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE): Proteins are separated based on the differences in their molecular weight.
4. Visualisation: Visualization of proteins by silver or coomassie brilliant blue staining.
5. Determination: Determination of molecular weight, pl, amount and others results by gel image analysis and mass spectrometry sequence analysis combined with database comparison.

• Isoelectric focusing and gel electrophoresis apparatus
• Sample solution
• Buffer solution and SDS-polyacrylamide gel