Blotted protein

In the early 1980s a serendipitous discovery was made when to a mixture of HRP, luminol, and hydrogen peroxide in buffer, firefly luciferin was accidentally added. The light yield of the HRP-catalyzed peroxidation of luminol was greatly enhanced. This remarkable discovery marked the beginning of a very successful analytical tool for immunoassay and all kinds of blotting (protein, DNA, and RNA) applications [16-18],... 

Proteins bound to the surfaces of synthetic membranes retain their antigenicity and are accessible to antibody probes. The most common membrane-based immunoassay technique is called immunoblotting or, more popularly, Western blotting. In Western blotting, proteins are transferred from an electrophoresis gel to a... 

Figure 8.11 Specific enzymatic immunodetection of a blotted protein. Depicted are blocked binding sites on the membrane (1), a primary antibody (2) specifically bound to an antigenic protein, and a secondary antibody (3) bound to the primary antibody. The secondary antibody is conjugated to a reporter enzyme (4). Substrate (S) is converted to insoluble product (P) at the site of the antigen.

Epitopes recognized by the antibody may become buried or denatured when the protein is transferred to surfaces such as nitrocellulose. This effect may be enhanced when blotting protein from SDS-containing gels. Insufficient contact between the gel and the membrane during transfer (e.g. air bubbles) will also reduce the amount of antigen available for recognition... 

For far Western blotting, native or recombinant proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to the nitrocellulose membranes and the blotted proteins are... 

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

Incubation of blotted proteins with antibody dilutions should not last less than 30 min and not more than 2 h at RT or may be kept in the refrigerator overnight. 

Anal Biochem 156 147). If antibodies are used raised against whole proteins, this thermal denaturation mostly destroys the epitopes on blotted proteins. 

Western blot Protein Yts Measures protein amounts... 

NHS esters of D-biotin have been used in many applications, including the biotinylation of rat IgE to study receptors on murine lymphocytes (Lee and Conrad, 1984), in the development of an immunochemical assay for a postsynaptic protein and its receptor (LaRochelle and Froehner, 1986a), in the study of plasma membrane domains by biotinylation of cell surface proteins in Dictyostelium disoideum amoebas (Ingalls et al., 1986), and for the detection of blotted proteins on nitrocellulose membranes after transfer from polyacrylamide electrophoresis gels (LaRochelle and Froehner, 1986b). 

Rat mouse brain SDS-PAGE LC-MS/MS Western blot Protein ID (Jordan et al. 2004)... 

The transfer of proteins from gels to nitrocellulose or other immobilizing matrices has become increasingly popular as a powerful tool for the subsequent analysis of proteins. Most frequently, the blotted proteins are analyzed for their antigenic properties by Western blotting. 

Figure 4.36. Western Blotting. Proteins on an SDS-polyacrylamide gel are transferred to a polymer sheet and stained with radioactive antibody. A band corresponding to the protein to which the antibody binds appears in the autoradiogram.

A Strategy to Obtain Internal Sequence Information from Blotted Proteins after Initial N-terminal Sequencing... 

Carbonic anhydrase and transferrin (60 picomole each) were run on a 10-20% SDS-polyacrylamide Tris-Tricine gel (1) and then electrically transferred to PVDF membrane in CAPS buffer (2). Tlie blotted proteins were stained with Coomassie Blue G-250 and the stained bands were excised for further study. 

Internal Sequence Information from Blotted Proteins... 

---