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.
- 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 untargeted separation of proteins.
- 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.
- We believe that the most effective method for detecting integrally bound membrane proteins is to isolate membranes from tissue, dissolve the membrane in Laemmli buffer and run on a 1-D gel. See the protocol Preparation of Soluble and Membrane Protein Fractions for details. A band or bands on the stained 1-D gel are cut out, digested with trypsin and analyzed by LC-MS/MS.
- Optimizing Sample Preparation for 2-D Gel Electrophoresis
- Protocol: Choice of Rehydration Buffers for 2-D Electrophoresis
- Protocol: Preparation of Soluble and Membrane Protein Fractions
- Protocol: Preparation of Plant Protein Extracts
- EZQ Protein Assay
- Protocol: Protease and Phosphatase Inhibitors
- Protocol: Use of PNGase F and Other Glycosidases
- Protocol Acetone Precipitation
- Protocol: Precipitation with Trichloroacetic Acid in Acetone
- Protocol: Chloroform/Methanol Precipitation
- Protocol: Phenol/Ammonium Acetate-Methanol Precipitation