Detection of Glycoproteins

Proteins with limited glycosylation can be detected following gel electrophoresis with the general protein stains such as CBB R-250 and silver. However, such staining gives no direct indication that these proteins are glycosylated and the methods are much less sensitive if the proteins are more highly glycosylated. Pro-teoglycans are usually stained with cationic dyes, such as Alcian Blue or Toluidine Blue, which bind to the negatively charged glycosaminoglycan side chains. Glycoproteins have generally been detected using variations of the Schiff base reaction, involving oxidation with periodic acid followed by staining with Schiff reagent, Alcian Blue or a hydrazine derivative. A twofold increase in sensitivity can be achieved with methods in which Alcian Blue is used as the primary staining agent followed by subsequent enhancement using a neutral silver-staining protocol.

An alternative approach to the analysis of glycosylated proteins is to radiolabel then in vitro, followed by gel electrophoretic separation of the radiolabelled proteins and their detection. N-linked sugar labelling can be achieved using [3H]-mannose and terminal O-linked N-acetylglucosamine can be labelled by galactosyltransferase and UDP-[3H]-galactose.

Probably the most versatile reagents for the characterization of glycosylated proteins following their separation by electrophoresis are radiolabelled, fiuor-escent or enzyme-conjugated lectins. Although it is possible to use these directly in the gel matrix, much better results are achieved using Western blotting techniques.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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