Detection of Proteins on Blot Membranes

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes (unitb3.2). PVDF is the preferred, more universal membrane and is emphasized here however, most stains work similarly on nitrocellulose, and many can be used on alternative blotting membranes. 

Stain Minimum amount detected Membrane type  

Protein sample electroblotted to PVDF, nitrocellulose, or nylon membrane 

Amido black 10B stain 0.1% (w/v) amido black (naphthol blue black 10B, Sigma) in 10% (v/v) acetic acid 5% (v/v) acetic acid Plastic boxes 

Place blot transfer membrane in a plastic box and wash with water three times for 5 min each. 

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Ruthenium complexes have been applied successfully to the luminescent detection of proteins on blotting membranes like nitrocellulose [160]. The bipyridyl and phenanthroline complexes modified with aminoreactive NHS-ester or isothiocyanate groups are commercially available [161]. An even higher sensitivity and lower detection limit can be obtained by encapsulating... 

Aoki, Y., Kunimoto, M., Shibata, Y., and Suzuki, K. T. (1986) Detection of metallothionein on nitrocellulose membrane using Western blotting technique and its application to identification of cadmium-binding proteins. Anal. Biochem. 157, 117-122. 

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). 

Ohtsu, L, Nakanisi, T, Furuta, M., Ando, E., and Nishimura, O., Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric identification of proteins on membrane detected by Western blotting and lectin blotting, Journal ofProteome Research, 4, 1391-1396, 2005. 

Tissue printing (pressure) This procedme allows the transfer of proteins to nitrocellulose membranes when a fresh cut organ or the cross sections of seeds, stems, tubers, leaves, and fruits are pressed against the membrane surface. This procedure was further modified to detect plant virus coat proteins on whole leaf blots. In this procedure, plant leaves to be blotted were placed on dry nitrocellulose membrane. The leaf and the nitrocellulose membrane were sandwiched on either side with blotting paper or one 0.75 inch thick block of plywood. The sandwich was positioned into a Carver laboratory press, and 10 000 psi of pressure was applied for 2 min. After disassembly, the membrane was air-dried for 20 min prior to standard immunodetection procedures. This procedure was shown to transfer protein uniformly. Some amount of chlorophyll was found to be transferred along with the protein, but this was not found to interfere significantly with the subsequent visualization of results. 

The specific labeled separated protein fractions blotted on a nitrocellulose membrane or specific immunoflxation-separated protein fractions in polyacrylamide after isoelectric focusing make it possible to detect some additional bands in CSF, i.e., IgM, IgA, free kappa or lambda light chains of specific antibodies (i.e., antiherpes, anti-borrelia, or anti-HIV) (LI, M3). 

The binding of an antibody to its antigen has also been utilized in the detection of specific proteins on a solid support, such as a western blot, where size-separated proteins are immobilized on a nitrocellulose or nylon membrane (see Section V.B). In this case, the primary... 

Specific detection of nitrocellulose membrane-bound proteins using a conjugated enzyme. (1) Proteins are transferred from electrophoresis gel to nitrocellulose membrane. Blocker proteins bind to unoccupied sites on the membrane. (2) The membrane is incubated with a primary antibody directed against the protein of interest. (3) A secondary antibody is directed against the primary antibody. (4) The second antibody is conjugated with an enzyme to provide a detection mechanism. Substrate solution is added to the blot. The conjugated enzyme (HRP or AP) catalyzes the conversion of substrate (S) to product (P) to form a colored precipitate at the site of the protein-antibody complex. 

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