| Assay Method Information | |
| | DELFIA |
| Description: | One hundred microliters of a 600 ng/mL solution of biotin-BID (Biotin-lc-EDIIRNIARHLAQVGDSMDR-NH2, where lc indicates a hydrocarbon chain of six methylene groups, aminohexanoic acid) was added to each well of 96-well streptavidin-coated plates (PerkinElmer). After 2 h of incubation and elimination by three washing steps of the unbound biotin-BID peptide, 11 μL of apreincubated solution of the protein and a serial dilution of the test peptide were added to the assay plate, followed by the addition of 89 μL of a 1.56 nM solution of Eu-N1-labeled anti-6xHis Antibody (PerkinElmer). After 1 h of incubation at RT to allow the immunoreaction to complete, a second washing step allowed removal of the unbound protein in complex with Eu antibodies if displaced by a test compound. Subsequently, 200 μL of enhancement solution (PerkinElmer) was added to each well and fluorescence measured after 10 min of incubation (excitation wavelength, 340 nm; emission wavelength, 615 nm). The protein final concentrations, determined by titrations, were 15 nM for hBfl-1, 16 nM for hMcl-1, 7 nM for hBcl-2, and 8.5 nM for hBcl-xL, while the antibody final concentration was 22.2 ng/well when compounds were tested against hBfl-1, hMcl-1, and hBcl-xL and 14.8 ng/well in the presence of hBcl-2. Each well received a final DMSO concentration equal to 1%. Protein, peptide, and antibody solutions were prepared in DELFIA assay buffer (PerkinElmer). |
| Affinity data for this assay | |
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