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Optimizing Sample Preparation for 2-D Gel Electrophoresis

Two-dimensional gel electrophoresis is a powerful technique for separating and visualizing proteins in a biological sample.

Despite many advances in alternative techniques, such as 2-D HLPC, it is still the most commonly used proteomics separation method. However, it is not a perfect technique for all proteins. Very large or hydrophobic proteins or proteins present in very minute quantities are often not seen on gels. These can be the proteins that are most interesting. Various approaches can be used to aid in the detection of these proteins.

1. Fractionate whole cell populations. If a sample is freed of highly abundant soluble or structural proteins, many proteins of lesser abundance can be detected. This can be done by separating proteins into soluble and membrane-bound populations, separating organelles, prefractionating on the basis of pI, or using any classic biochemical method for separation of proteins.
2. Use alternative methods for IEF. The standard method for 2-D electrophoresis uses immobilized pH gradient (IPG) strips for isoelectric focusing. While these strips yield extremely reproducible results for the first dimension, they are a problem for very large or hydrophobic proteins. Such proteins either do not absorb into the strips or precipitate after absorption. As an alternative, isoelectric focusing can be performed by a solution method using our Bio-Rad MicroRotofor. Fractions produced by this method are then applied to large pore 1-D gels. Large membrane proteins are often visible by this method.

• Protocol: Explanation of Our Sample Preparation Alternatives
• EZQ Protein Assay
• Protocol: Isolation of Membrane Proteins
• Protocol: Reduction and Alkylation Before First Dimension Protocol
• Protocol Acetone Precipitation
• Protocol: Precipitation with Trichloroacetic Acid in Acetone
• Protocol: Chloroform/Methanol Precipitation
• Protocol: Phenol/Ammonium Acetate-Methanol Precipitation