Notes and TipsComparison of Three Chemiluminescent Horseradish Peroxidase Substrates for Immunoblotting
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Chapter 33 Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection
2009, Methods in EnzymologyCitation Excerpt :The most significant advances in Western blotting methodology are highly sensitive-enhanced chemiluminescent substrates, imaging systems, and, most recently, the availability of a wide variety of photostable fluorophores. The widespread use of extremely sensitive chemiluminescent substrates (Mattson and Bellehumeur, 1996; Walker et al., 1995) has resulted in nearly eliminating the use of radioisotope-labeled probes. Protein A or G labeled with 125I was once commonly used as a secondary detection reagent; however, the enhanced chemiluminescent substrates can detect proteins down to the low-femtogram level with high signal-to-noise ratios.
Blocking tumor necrosis factor-α inhibits folic acid-induced acute renal failure
2006, Experimental and Molecular PathologyGenetic heterogeneity of the MYC oncogene in advanced juvenile angiofibromas
2006, Cancer Genetics and CytogeneticsCitation Excerpt :For sodium dodecylsulfate gel electrophoresis, 150 μg protein was placed per lane and Western blot was performed as previously described [21]. For antigen detection, a monoclonal C-MYC antibody (1:500, DAKO, Glostrup, Denmark) was used and visualized by a corresponding POD-marked secondary antibody (Dianova, Dörentrup, Germany) followed by the lumi-light method according to Mattson and Bellehumeur [22]. For immunohistochemical staining, 6-μm juvenile angiofibroma sections were deparaffinized.
Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli
2002, Journal of Biological ChemistryCitation Excerpt :Horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were used as secondary antibodies for the His and T7 antibodies, respectively. Immunocomplexes were revealed with an ECL kit according to the manufacturer's protocol (30). SDS-PAGE (12%) was carried out according to the method of Laemmli (31).
Two Forms of UvrC Protein with Different Double-stranded DNA Binding Affinities
2001, Journal of Biological ChemistryCitation Excerpt :7 or 10% acrylamide, 0.32% bisacrylamide, and SDS were used as described by Laemmli (23). After electrophoresis the proteins were electrotransferred to a nitrocellulose membrane and reacted with polyclonal UvrC antiserum (18); the antigen-antibody complex was further conjugated with horseradish peroxidase-labeled antibodies and then detected by chemiluminescence (24). Several purification schemes have been reported for UvrC purification, and they have in common two steps: phosphocellulose and ss-DNA-cellulose chromatography (15, 25, 26).