![]() ![]() Cross-reactivity of the secondary antibody is also eliminated. Fewer steps are required making it considerably faster than other types of ELISA. Direct ELISA is considered to be the simplest type of ELISA. Direct ELISA is suitable for determining the amount of high molecular weight antigens. This correlation can be used to extrapolate the concentration of antigen in an unknown sample from a standard curve. The appropriate substrate is then added to the medium, producing a signal directly proportional to the amount of antigen in the sample. Incubation is followed by washing, which removes the unbound antibodies from the medium. An enzyme-tagged antibody enables its measurement. The surface of the plate is coated directly with the sample. This was the ELISA originally developed by Perlmann and Engvall. Below, we have highlighted commonly used ELISA assays and their use to determine which is suitable for your research needs. Enzo Life Sciences’ catalog of nearly 300 ELISA kits includes sensitive, specific, and reliable assays for relevant markers of bioprocess, heat shock response, inflammation and immune response, oxidative stress, signaling pathways, steroid and peptide hormones, and much more. The crucial component of ELISA-based detection and quantification is the specificity of the interaction between the marker of interest and the antibody. The essential components of ELISA are threefold: an antigen to detect and perhaps quantitate, a specific antibody to this antigen, and a system to measure the amount of antigen in a given sample. Drug treatment correlated with the induction of autophagy as indicated by the decrease in p62 levels (A and B) and by elevation of LC3-II levels (C). Cells were harvested 6 and 12 hours post-treatment, lysed, and analyzed with our p62 ELISA kit, and for p62 and LC3-II by Western blot. MDA-MB-231 human breast cancer cells were treated with 2µM of witharefin A (WA), an autophagy-inducing drug or vehicle. Below is an example of how a p62 ELISA kit can be used to monitor the induction of macroautophagy. ELISA can also serve as an orthogonal assay format to other methods such as activity assays or Western blotting to further support and enhance previously reported research results. ![]()
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