Serum Glycoproteins by SDS-PAGE and Western Blotting

Identification of Serum Glycoproteins by SDS-PAGE and Western Blotting 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

In Experiment 4, your sample of a-lactalbumin extracted from bovine milk was subjected along with other proteins to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After staining with the dye Coomassie Blue, deeply colored bands appeared on the gel wherever there was a protein. You suspected that some of the blue bands on the gel were due to a-lactalbumin. If molecular weight standards were included on the slab gel, you were able to estimate the molecular weight for a-lactalbumin and other proteins. SDS-PAGE is indeed a very effective analytical tool to achieve fractionation of protein mixtures, to analyze purity, and to estimate molecular weight, but it provides no experimental data to prove the identity 

The actual blotting process may be accomplished by one of two methods passive (or capillary) transfer and electroblotting. In passive transfer, the membrane is placed in direct contact with the polyacrylamide gel and organized in a sandwich-like arrangement consisting of (from bottom to top) filter paper soaked with transfer buffer, gel, membrane, and more filter paper. The sandwich is compressed by a heavy weight. Buffer passes by capillary ac- 

---