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Polysorbate 20 is also known as Tween 20, a commercial brand name. It is a common detergent used in many buffers for washing nitrocellulose membrane in western blotting and microtiter plate wells in ELISA assays. Tris is a buffer that maintains a pH of 7–9.2.
[28] [29] ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs. Enzyme-linked immunosorbent assay plate. The ELISA was the first screening test widely used for HIV because of its high sensitivity. In an ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV antigens are attached.
3,3′,5,5′-Tetramethylbenzidine or TMB is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualising reagent used in enzyme-linked immunosorbent assays (). [1]
After blocking, a solution of primary antibody (generally between 0.5 and 5 micrograms/mL) diluted in either PBS or TBST wash buffer is incubated with the membrane under gentle agitation for typically an hour at room temperature, or overnight at 4°C. It can also be incubated at different temperatures, with lesser temperatures being associated ...
One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining.
The antibody that fails to react is known as the blocking antibody and prevents the precipitating antibody from binding to the antigens. Thus the proper precipitation reaction does not take place. However, when the serum is diluted, the blocking antibody is as well and its concentration decreases enough for the proper precipitation reaction to ...
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