| Assay Method Information | |
| | BCL6 TR-FRET Protocol |
| Description: | Assay buffer A: 50 mM HEPES pH 7.5, 125 mM NaCl, 0.01% TritonX.Assay buffer B (made fresh): buffer A+1 mM Glutathione (or 0.5 mM DTT).Assay buffer C (made fresh): buffer B+0.03% BSA.Black Proxy plates, 96 well.15 μl final reaction volumes (BCoR-Cy5 100 nM, SA-Eu 2 nM, BCL6-avitag 2 nM).134 μM BCL6-Avitag-Biotin stock: made fresh by adding 2 μl of BCL6-Avitag-Biotin to 31.5 ml Buffer C.1 mM BCoR-Cy5 peptide (LifeTein) stock in Dimethylformamide (DMF).300 nM BCoR-Cy5 working stock: made fresh by adding 4.5 μl of the 1 mM BCoR-Cy5 peptide stock to 15 ml Buffer B.10 μM Eu-Streptavidin (Lance Eu-W1024 Streptavidin, PerkinElmer) stock solution.6 nM Eu-Streptavidin working stock: made fresh by adding 9 μl Eu-Streptavidin stock solution to 15 ml Buffer A.Compounds were diluted to 10 mM. Twenty microliters of DMSO was aliquoted to each well of the microtiter plates. From the 10 mM compound stock, 8.7 ul was aliquoted to the 20 ul DMSO and 3-fold serial dilutions (12 pt 3-0.01 uM tritration plate, 96 well, 100% DMSO) performed. Five ul from the titration plate wells was aliquoted to 45 ul Buff C (Intermediate dilution plates, 10% DMSO).Spot 1.5 μl compound titrations to 384-well plates in duplicate, and spot 3.5 μl [8.5 nM] BCL6-bio protein to each well. The plate was mix briefly, centrifuged, and incubated for 30 minutes at room temperature.Mix 14 mls of BCoR-Cy5 [300 nM] and 14 mls [6 nM] Eu-Streptavidin. Spot 10 μl BCoR-Cy5/SA-Eu (1:1) mix to each well. The plates were incubated for 2 hours and then read on an Envision plate reader. |
| Affinity data for this assay | |
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