The analysis of glycoproteins in cells and tissues by two‐dimensional polyacrylamide gel electrophoresis

Abstract

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.

Differential Expression of Serum Clusterin Isoforms in Colorectal Cancer

“…It was also established that the synthesis of Nglycans with poly-N-acetyllactosamine side chains is associated with tumor cells showing high metastatic capacity compared with those with low metastatic potential (54). It is also a fact that an aberrant glycosylation decreases the affinity of an oligosaccharide to the Con A (46,(55)(56)(57). Hence should it be due to an increased sialylation or to a change in structure, the fact is that the CLU isoform eluted in FI does not bind to the Con A matrix and specifically appears in relation to the CRC disease.…”

Fluorescence Detection of Glycoproteins in Gels and on Electroblots

“…The analysis of protein glycosylation is readily accomplished by polyacrylamide gel electrophoresis (Koch and Smith, ; Packer et al, ; Taverna et al, ; Koketsu and Linhardt, ). However, relatively few high‐sensitivity methods have been developed to reliably detect oligosaccharide residues covalently attached to proteins for visualization in polyacrylamide gels or on electroblot membranes (Packer et al, ; Packer et al, ; Koketsu and Lindhardt, ).…”

A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype

“…Sorcin [260] is an EF-hand Caz'-binding protein of unknown function that is overexpressed in multidmgresistant cells [261] and that has been involved in the development of the multidrug resistance phenomenon [261,2621. The implications of the increased expression of this protein in transformed K14 keratinocytes ( Fig.…”