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.
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.