Protein, separation by electrophoresis

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

The antigen must be in a highly aggregated or immobilized form. This technique is frequently used to produce antibodies to proteins separated by electrophoresis. The gel is stained in the normal manner and the proteins blotted over onto nitrocellulose. The band containing the protein of interest is then excised from the blot and used... 

CIO. Crook, E. M., Harris, H., and Warren, F. L., Continuous direct photometry of proteins separated by electrophoresis in filter paper. Biochem. J. 51, Proc. xxvi (1952). 

Some of the most sensitive procedures for detecting proteins separated by electrophoresis are based on immunological methods. After electrophoresis, proteins are transferred by blotting on to a membrane such as PVDF (Timmons and Dunbar 1990), and then detected by methods similar in principle to those described for the ELISA experiments. 

Q.26.13 What factors impact protein separation by electrophoresis What does a scientist typically do if they want to separate proteins by molecular weight only ... 

A very useful method to produce antibodies against proteins separated by electrophoresis and stained with CBB has been reported by Boulard and Lecroisey (1982). In this method (Section 5.2.1)... 

A. V. Lemmo and J. W. Jorgenson, Two-dimensional protein separation by mictocolumn size-exclusion chromatography-capillary zone electrophoresis , 7. Chromatogr. 633 213-220(1993). 

Figure 26.2 Separation of a protein mixture by electrophoresis. At pH 6.00, a neutral protein does not migrate, a basic protein is protonated and migrates toward the negative electrode, and an acidic protein is deprotonated and migrates toward the positive electrode.

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

Various methods have been used to examine the composition of proteins adsorbed to SAMs. Overall adsorption patterns can be examined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [50, 76, 77]. Absorbed proteins are eluted from the surface with surfactant (SDS), and then separated by electrophoresis. The proteins of interest are examined by western blotting [50, 76, 77]. Protein-specific antibodies can be used to detect proteins of... 

The 5-iodoacetamido derivative of fluorescein (5-IAF) has been used to label numerous proteins and other biomolecules, including actin (Plank and Ware, 1987), myosin (Aguirre et al., 1986), troponin (Greene, 1986), hemoglobin (Hirsch et al., 1986), and sulfhydryl-containing proteins separated by SDS electrophoresis (Gorman, 1984). 

Yan, J. X. Harry, R. A. Spibey, C. Dunn, M. J. Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 2000, 27(17), 3657—3665. 

Lahm, H.W., Langen, H. (2000). Mass spectrometry A tool for the identification of proteins separated by gels. Electrophoresis, 21, 2105-2114. 

Western blots Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen. 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

N. E. Baryla, J. E. Melanson, M. T. McDermott, and C. A. Lucy, Characterization of Surfactant Coatings in Capillary Electrophoresis by Atomic Force Microscopy, Anal. Chem. 2001, 73, 4558 M. M. Yassine and C. A. Lucy, Enhanced Stability Self-Assembled Coatings for Protein Separations by Capillary Zone Electrophoresis Through the Use of Long-Chained Surfactants, Anal. Chem. 2005, 77, 62. 

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