| Determination of purity and molecular weight of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
|
| HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) |
HPLC is a powerful chromatographic technique for high-resolution separation of proteins, peptides, and amino acids . The principle of the separation may be based on the charge, size, or hydrophobicity of proteins. The narrow columns are packed with a noncompressible matrix of fine beads coated with a thin layer of a stationary phase. A protein mixture is applied to the column, and then the components are eluted by either isocratic or gradient chromatography. The eluates are monitored by ultraviolet absorption, refractive index, or fluorescence. This technique gives high-resolution separation with high specificity and high sensitivity and is the most common technique for purification of proteins and peptides. |
| Electrophoresis can be used for the separation of a wide variety of charged molecules, including amino acids, polypeptides, proteins, and DNA. When a current is applied to molecules in dilute buffers, those with a net negative charge at the selected pH migrate toward the anode
and those with a net positive charge toward the cathode. A porous support, such as paper, cellulose acetate, or polymeric gel, is commonly used to minimize diffusion and convection.
|
| Like chromatography, electrophoresis may be used for preparative fractionation of proteins at physiologic pH. Different soluble proteins will move at different rates in the electrical field, depending on their charge to mass ratio. A denaturing detergent, sodium dodecylsulfate (SDS), is commonly used in a polyacrylamide gel electrophoresis (PAGE) system to separate and resolve protein subunits according to molecular weight. The protein preparation is usually treated with both SDS and a thiol reagent, such as β-mercaptoethanol, to reduce disulfide bonds. Because the binding of SDS is proportional to the length of the peptide chain, each protein molecule has the same mass-to-charge ratio and the relative mobility of the protein is proportional to the molecular mass of the polypeptide chain. Varying the state of crosslinking of the polyacrylamide gel provides selectivity for proteins of different molecular weights. A purified protein preparation can be readily analyzed for homogeneity on SDS-PAGE by staining with sensitive and specific dyes such as Coomassie Blue, or with a Silver staining technique, as shown in Figure 2.15.
|
|
