| Assay Method Information | |
| | Fluorescence Polarization Assay |
| Description: | The protocol for the CBX7 Fluorescence Polarization assay is as follows: A standard FP buffer (20 mM Tris, 250 mM NaCl, 0.01% w/v Tween20) at pH 8.0 is created and make up one stock solution at 1× final concentration in the FP buffer. The solution contains Human His6-CBX7 used with FITC-VARKme3SA, at 250 nM and 10 nM respectively. For 384 well assay formats, 10 uL of solution is added to each well of the assay plate and the plate is spun at 1000 rpm for 30 seconds. 100 nL of experimental compounds from stock plates are delivered by robotic pin transfer using a Janus Workstation (PerkinElmer), allowing the compounds to interact with CBX7 binding prior to assay measurement, followed by another spin and an incubation room temperature. Fluorescence Polarization measurements are performed on an Envision 2104 (PerkinElmer) utilizing the manufacturer's protocol that has the correct excitation and emission wavelengths, cutoff filters, delay time, etc. A crosstalk calculation is also done through the Envision software to correct for luminescence for adjacent wells while reading the plate. |
| Affinity data for this assay | |
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