Western blot enhanced chemiluminescence

There are several advantages to performing the denaturation, renaturation, and kinase procedures with the proteins electroblotted onto a membrane rather than with the SDS-polyacrylamide gel. First, after phosphorylation, the same blot can be used for immunoblotting depending on the antibody and the integrity of the epitope recognized. We were able to successfully use monoclonal antibodies to CaM kinase a and / and the Amersham Enhanced Chemiluminescence (ECL) Western blotting detection system to detect the isoforms after in situ renaturation (Shackel-... 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Constantine, N. T. Bansal, J. Zhang, X. Hyams, . C. Hayes, C. Enhanced chemiluminescence as a means of increasing the sensitivity of western blot assays for HIV antibody. J. Virol. Methods 1994, 47(1-2), 153-164. 

Wigle, D. A., Radakovic, N. N., Venance, S. L., and Pang, S. C. 1993. Enhanced chemiluminescence with catalyzed reporter deposition for increasing the sensitivity of western blotting. Biotechniques 74 562-563. 

Other applications of this type of detection include Western blotting procedures for the identification and visualization of protein antigens (LI, L8, L9). One such recent assay for herpes simplex virus Type 2 showed that enhanced chemiluminescence is 500 times more sensitive than a colorimetric method (Dl). Antibodies to several of the HIV viral proteins have also been detected in chemiluminescent Western blots (SI6). 

Smith, H. V., Mayambo, T. M., Dunlop, E. M., and Holmes, P. H., Analysis of drug binding to serum macromolecules using a biotin-strepatvidin enhanced chemiluminescent western blot technique. In Bioiuminescence and Chemiluminescence Current Status (P. E. Stanley and... 

Fig. 5. ADP-ribosylation of RhoA by DC3B inhibits GTPyS-induced RhoA translocation from the cytosolic to the membrane fraction. After incubation with or without the chimeric toxin DC3B (48 hr), a-toxin-permeabilized tissues were stimulated with GTPyS (50 pM) for 20 min and homogenized, fractionated and separated into cytosolic (C) and Triton-extracted particulate fraction (P) translocation of RhoA to the membrane fraction was inhibited by DC3B treatment (48 hr). A Representative Western blots of RhoA visualized by enhanced chemiluminescence. Arrows indicate position of mol wt markers remainder of gels did not show other bands and are not shown. B Summary of the effect of DC3B on translocation of RhoA by GTPyS (50 xM) from the cytosolic to the membrane fraction. DC3B significantlyinhibited translocation of RhoA (from Fujihara et al. 1997).

Huang, D. Amero, S. A. Measurement of antigen by enhanced chemiluminescent western blot. Biotechniques 1997,22(3), 454-456,458. 

Total mouse liver proteins (50 were separated on a 10% sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) and Western blotting was performed by electrophoretic transfer of the separated proteins onto a nitrocellulose filter (Nitropure, Micron Separations Inc., Westborough, MA, USA) using a Mini-Protean II device (Bio-Rad). The blots were probed with an anti-MTE-I antibody and subsequently with horseradish peroxidase-conjugated secondary antibodies and visuahsed by enhanced chemiluminescence (ECL, Amersham) using X-ray film. 

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