Western blot transfer procedure

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 the Eastern-Western procedure, lipids are first applied to a solid support such as nitrocellulose. Next, proteins subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis are transferred by standard Western blotting techniques to the solid support in such a manner that the protein of interest is transferred to the lipid patch. During electrotransfer of protein to the solid support, protein, lipid, and sodium dodecyl sulfate mix and as transfer continues the sodium dodecyl sulfate is removed leaving behind the protein to refold in the presence of lipid. Attachment of the refolded protein to a solid support allows one to probe protein structure using conformation-sensitive antibodies or protein function... 

Procedures for SDS-PAGE, transfer to nitrocellulose membranes and western blot are described elsewhere (26), and require no major modifications for chemokine receptor analysis (rccNote 4). 

H. Towbin, T. Staehelin, and J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1979) A. Klapper, B. MacKay, and M. D. Rash, Rapid high resolution Western blotting from gel to image in a single day, BioTechniques 12, 650-654 (1992). 

For western blotting procedure the proteins were transferred from the gels to nitrocellulose papers by elektro transfer with a semi dry blotting system. Immunedetection of ELIP was carried out in general as described by Towbin et al (8) with a horse-radish-peroxidase-conjugated secondary antibody to visualize the antigen-antibody-complex on the nitrocellulose sheets. 

Both universal staining procedures and specific detection techniques can be performed after (electro) transfer or (electro)blotting of proteins (also called Western blotting) from the gel matrix (which sometimes hinders protein analysis) to a nitrocellulose or polyvinylidenedifluoride membrane, to which they are bound and immobilized. On the membrane, protein molecules are faster and better accessible for the interactions with the applied specific antibodies. The antigen-antibody complexes are visualized by a second antibody (against the first antibody) with an attached enzyme label, which catalyzes the color reaction in the place of the protein zone. 

This method involves electrophoretically separating the protein fraction of CS fluid and subsequently transferring these proteins to a nitrocellulose membrane according to Western blot procedures. After saturating the free binding sites on the membrane with a Tris-buffered salt solution, the membrane is treated with the 14-3-3-specific antibody anti-14-3-3 polyclonal rabbit antibody . A second antibody binds this antibody, the alkaline phosphatase-conjugated anti-rabbit IgG antibody . Detection is achieved by a colorimetric reaction. 

Transfer the proteins after SDS-PAGE to a polyvinylidene fluoride (PVDF) membrane and detect the proteins by specific antibodies at 1 2,000 dilutions using Western blot procedures, or by Coomassie Blue staining. Alternatively the proteins can be identified by mass spectrometry as will be described in the following paragraph. 

PSQ, 0.1 j,m pore size, Millipore, USA) and enclosed in a Western Blot cassette. After electro-transfer, 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 Coomassie Brilliant Blue solution. A detailed description of this procedure can be found in [369, 370). 

There are three basic steps to the western blot procedure separation of proteins by gel electrophoresis, transfer of the separated samples from the gel to a membrane, and probing the membrane with antibodies. Proteins carry highly variable charges which are not easy to separate by simple gel electrophoresis. Therefore, sodium dodecyl sulfate (SDS) is often applied to denature the proteins and apply a uniform negative charge concentration to all of the polypeptides. The polypeptides can then be separated via polyacrylamide gel electrophoresis according to size. 

The main procedures of western blotting are similar to those of Southern and northern blotting, including gel electrophoresis, transfer to a membrane, and probe hybridization. However, there are differences of particular note ... 

The electrotransfer of proteins onto (non-specific) binding membranous sheets is named Western hlot in contrast to the transfer of DNA (Southern hlot) and of RNA (Northern hlot). The main advantage of blotting procedures lies in the immobilization and presentation of macromolecules on the surface of a solid planar material. This presentation leads to an easy access of reactants in the opposite to the diffusion-controlled motion of reaction partners within gels or macroporous spheres. 

This procedure is similar in principle to Southern and Northern blots, but it is designed for the transfer of proteins from gels onto nitrocellulose membranes. An electrophoretic technique is often used to speed the transfer by 10-fold, and the apparatus used for such Western transfers is shown in Figure 9.15. The rapid electrophoretic process ensures that complete transfer occurs with minimal diffusional zone broadening. 

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