Oligosaccharide Analysis of Membrane Coupled Glycoproteins

In this section, the determination of glyco moieties of a special class of glycoproteins, the class G immunoglobulins (IgG), is described. Immunoglobulins G consist of two heavy and two light polypeptide chains and carry their carbohydrates mainly in the conservative region of the heavy chains (Fc). Additionally, 

Separation and Purification of IgC via SD5-PACE (SD5 Polyacrylaniide Cel Electrophoresis) and Electro-Transfer of Immunoglobulins onto an Inert PVDF Membrane . 

About 50 pg of a glycoprotein are mixed with the same volume of a double concentrated SDS buffer and denatured at 100 °C. The samples are applied onto the prepared SDS polyacrylamide gel, concentrated electrophoretically in the collecting gel, and separated to purity in the separation gel. After this step, the separation gel is put onto a previously activated PVDF membrane (Immobilon 

2) PVDF is supposed to be inert and does not bind carbohydrates [366]. 

m pore size, Millipore, USA) and enclosed in a Western Blot cassette. After electro-transfer, the protein on the membrane is stained with a 0.1% Coomassie Brilliant Blue solution. The membrane is washed several times to remove residues from the SDS gel electrophoresis. The completeness of the transfer can be checked by staining the gel in the Coomassie Brilliant Blue solution. A detailed description of this procedure can be found in [369, 370). 

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