Electrotransfer immunoblotting

Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting)... 

This chapter will describe in detail the procedure for Western blotting of polypeptides and proteins separated on a denaturing polyacrylamide gel system. This procedure is used routinely in our laboratory for the analysis of polypeptides from a variety of subcellular fractions of whole tissue and cell lines, and has evolved over a number of years in the hands of several people. Many different immunoblotting procedures are currendy available details of variadons from the I-protein-A method described here are given in the Notes secdon, as are brief amendments covering electrotransfer from two-dimensional and isoelectric focusing systems. A detailed descripdon of sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) is not appropriate for this chapter, and the reader is referred to vol. 1, Chapter 6, and refs. 9-14. For details of the producdon of polyclonal and monoclonal andsera, Chapters 1-6 in this vol. 

Optimization of the conditions during electrotransfer and immunoblotting is discussed in ref. 6. 

Separation of polypeptides by SDS-PAGE on mini gels 272 Electrotransfer of proteins from gels to nitrocellulose membranes 275 Staining membranes with Ponceau S after transfer to obtain the total protein pattern 276 Immunoblotting 278... 

FIGURE 9.3 Immunodetection of PrP and PrP in brain extracts. Western immunoblot of brain homogenates from iminfected and prion-infected animals (e.g., Syrian hamsters). Samples were either not digested (-) or digested (+) with proteinase K for 30 min at 37°C prior to polyacrylamide gel electrophoresis (PAGE). After electrotransfer, the blot is developed with a polyclonal rabbit anti-PrP antiserum that recognizes both PrP and PrP . PrP (blue band) is completely hydrolyzed under these conditions, whereas PrP (red band) is partially resistant to the protease treatment. In the third lane, the purple spot represents a mixture of PrP and PrP that are codetected in brain extracts from infected animals. 

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