Native PAGE
Overview
Native polyacrylamide gel electrophoresis is an electrophoretic separation method typically used in proteomics.
Native PAGE should not be confused with SDS-PAGE. SDS-PAGE uses a detergent, sodium dodecyl sulfate, to denature proteins. The primary effect is that proteins in protein complexes are separated, reducing the dependence of protein mobility on folding.
In contrast, native PAGE separations are run in non-denaturing conditions. Detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain--for the most part--associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, since they cannot move through the polyacrylamide gel as quickly as individual, denatured proteins.
There are three popular methods of native PAGE, clear native (CN-PAGE), blue native (BN-PAGE), and quantitative preparative native continuous (QPNC-PAGE).
Blue Native PAGE
BN-PAGE is more widely used than CN-PAGE, since the use of Coomassie blue dye enables the separations to be examined with the naked eye. The disadvantage of using dye is that in binding to proteins it causes complexes to dissociate.
Clear Native PAGE
CN-PAGE uses no dye, which makes it difficult to examine separations of proteins. However, this method is arguably the most useful for examining protein-protein interactions, particularly in conjunction with mass spectrometry (MS).
In a typical CN-PAGE experimental procedure, the complexes will be separated with CN-PAGE. An additional separation method may then be used, such as isoelectric focusing. The gel will then be physically cut, and the protein complexes extracted from each portion separately. Each extract may then be sequenced, such as by peptide mass fingerprinting. This can provide a great deal of information about the identifies of the proteins in a complex.
See also
External links
Discontinuous native protein gel electrophoresis Template:WH Template:WikiDoc Sources Template:Jb1