Histone H3 (H3) BioAssay™ ELISA Kit (Human, Mouse, Rat) is a sandwich enzyme immunoassay for the in vitro quantitative measurement of H3 in tissue homogenates, cell lysates and other biological fluids.
Test Principle:The microtiter plate provided in this kit has been pre-coated with an antibody specific to H3. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to H3. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain H3, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of H3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Storage and Stability:Store *025706A, *025706B, *025706D and *025706E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.
Assay Procedure Summary:1. Prepare all reagents, samples and standards
2. Add 100ul standard, control or sample to each well. Incubate 2 hours at 37°C
3. Add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C
4. Aspirate and wash 3 times
5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C
6. Aspirate and wash 5 times
7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37°C
8. Add 50ul Stop Solution. Read at 450nm immediately.
