Protein separation electrotransfer from gels

The transfer protocols are the same for DNA and RNA. In opposition to the methods given in Protocol 2.5.3, the forces driving the biomolecules from the separating gel to the receiving membrane are diffusion and capillary flow. This type of transfer is also applicable for proteins, but because the pores of polyacrylamide gels used for protein separation are mostly smaller, transfer times are longer and transfer efficiencies are lower than by electrotransfer. 

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. 

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 Inununoblotting 278... 

Identify the protein spots as usual, i.e., by staining, autoradiography, gel overlay, or Western blot. In the latter case the gel must be separated from the GelBond foil prior to electrotransfer. For this purpose a film remover (Gorg 2003, Fig. 19) is used The gel is placed on the cylindrical remover with foil down, clamped on an edge, and a thin stainless steel or nylon wire is pulled between foil and gel towards to your body. Cover the gel with the wetted blotting membrane (cf Protocol 2.4.3) and transfer membrane as well as gel to the blotting apparatus. 

Separation and Purification of IgG via SDS-PAGE and Electrotransfer of Immunoglobulines onto an Inert PVDF Membrane About 50 pg of a glycoprotein is 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 piuity in the separation gel. After this step, the separation gel is put onto a previously activated PVDF membrane (Immobilon PSQ, 0.1 pm pore size, Millipore, USA) and enclosed in a Western Blot cassette. After electrotransfer, 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... 

---