Enrichment and Purification of Proteins

Free-Flow Electrophoresis of Protein Samples To reduce the dynamic range of proteins to be analyzed, and subsequently to maximize the number of 

Two-Dimensional Gel Electmphoresis 2-DE is the most powerful and robust technique available to separate a complex mixture of proteins from biological specimens [13-15]. It is a two-step process in which each dimension makes use of complementary properties of proteins for their separation. The first dimension is usually performed in the lEF mode, and the second in the conventional, size-based separation mode. The lEF mode separates proteins on the basis of their inherent charge or isoelectric point (pi values). In the second dimension, proteins are separated on the basis of molecular size, typically by SDS-PAGE. With this orthogonal separation scheme, a mixture that contains thousands of proteins can be separated in a single experiment to provide a two-dimensional image. The following key steps are used in a typical 2-DE separation of proteins  

For the first-dimension separation, a protein sample containing an appropriate cocktail of carrier ampholyte is applied to a standard immobilized pH gradient gel (IPG) strip and left overnight to rehydrate the strip. These strips are available in several narrow pH ranges for improved separation and are specially well suited for FFE-separated subfractions. Application of the appropriate electrical potential to the rehydrated strip results in separation of the proteins according to their intrinsic charge. 

The focused strips are equilibrated with SDS-Tris buffer that also contains a reducing agent (e.g., 2-mercaptoethanol or dithiothreitol) and an alkylating agent (e.g., iodoacetamide) prior to second-dimension separation. 

The IPG strips after reduction and alkylation are placed at the top of the SDS polyacrylamide gel slab. 

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M Fountoulakis, H Langen, C Gray, B Takacs. Enrichment and purification of proteins of Haemophilus influenzae by chromatofocusing. J Chromatogr 806 279-291, 1998. 

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