| Assay Method Information | |
| | ERα TR-FRET Test |
| Description: | 1x Tris-HCl (Sigma, PHG0002) protein buffer was prepared and mixed for later use. The compound to be detected was prepared into a stock solution with a concentration of 2 mM and then subjected to serial 3-fold gradient dilution, resulting in a total of 10 concentrations. The diluted compounds were respectively added to a reaction plate by means of Echo 550, with 100 nL per well, and at the same time 5 nL of estradiol (Sigma, 491187; final concentration 1.5 nM) was added to each well.Preparation of 1x protein mixed solution: firstly, 2x GST-ERalpha-LBD (Invitrogen, A15677)/MAb anti-GST-Eu (Cisbio, 61GSTKLA) mixed solution was prepared according to the following table.Final Working Concentration ofconcentration concentration stock solutionSubstance (nM) (nM) (nM)GST-ERalpha-LBD 2 4 20100MAb anti-GST-Eu 2.5 ng/well 50 nl/well 50 ug/ml2x biotin-SRC2/streptavidin-XL665 (Cisbio, 610SAXLA) mixed solution was prepared.Final Working Concentration ofconcentration concentration stock solutionSubstance (nM) (nM) (nM)Biotin-SRC2 75 150 1000000Streptavidin-XL665 50 ng/well 50 nl/well 1 mg/mlThe above 2x GST-NR-LBD/ MAb anti-GST-Eu solution and 2x biotin-SRC2/streptavidin-XL665 solution were uniformly mixed at a volume ratio of 1:1; and the 1x protein mixed solution was added to each well of a 384-well plate, with 20 uL being added per well, the 384-well plate was put into a centrifuge and centrifuged at room temperature at 1000 rpm for 10 seconds, and it was taken out, then left to stand at room temperature for 3 h, and then read by EnVision multifunctional microplate reader.The values at 665 and 615 (nm) were read out, and with the value at 615 as the correction value, the final value was expressed as the value at 665/the value at 615. |
| Affinity data for this assay | |
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