The analysis of glycoproteins in cells and tissues by two-dimensional polyacrylamide gel electrophoresis.
Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I] concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combination of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
G L
GL
Koch
M J
MJ
Smith
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis; D002454:Cell Differentiation; D002460:Cell Line; D003599:Cytoskeleton / Q000032:analysis; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional; D004721:Endoplasmic Reticulum / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis*; D006649:Histocompatibility Antigens / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis
D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D006649:Histocompatibility Antigens; D008562:Membrane Glycoproteins