| Assay Method Information | |
| | AlphaLISA hTNFa kit |
| Description: | TNFα protein levels were determined using an AlphaLISA hTNFa kit (Perkin Elmer) according to the manufacturer's instructions. Briefly, the samples were allowed to return to room temperature and centrifuged at 1500×g for 5 min. Samples were diluted 1/5 (5 μL sample in 20 μL AlphaLISA buffer). At the same time, a standard curve of TNFα was prepared by serial 1/3 dilutions from a stock solution (5000-2 pg/mL). 5 μL sample/standard curve were transferred to a 384-well Optiplate , and to this was added 20 μL anti-humanTNFa acceptor beads/biotinylated antibody mix. The plate was incubated at room temperature for 60 min. After this incubation, 25 μL streptavidin donor beads were added, and the plate was incubated for a further 60 min in the dark at room temperature. The samples were read at 615 nm with excitation at 680 nm using an Envision plate reader. TNFα in the samples was determined by extrapolation from the standard curve and expressed as pg/mL.The % inhibition of TNFα was determined by the equation: % inhibition=(1−(A−B)/(C−B))×100 Here, A=TNFα in LPS stimulated samples containing compound, B=TNFα in unstimulated samples. and C=TNFα in LPS stimulated samples without compound. Percent inhibition was plotted against concentration, and a curve graphed using a 4-parameter curve fit (Xlfit 4.1) to determine the pIC50. |
| Affinity data for this assay | |
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