| Assay Method Information | |
| | EMSA assay for RPA-DNA inhibition activity |
| Description: | EMSA reactions (20 μL) were performed with 50 nM fl-RPA and 2.5 nM 5′[32P]-labeled 34-base DNA in buffer containing 20 mM HEPES (pH 7.0), 1 mM DTT, 0.001% NP-40, 100 mM NaCl, 5 mM MgCl2 and 50 μg/ml bovine serum albumin (BSA). Chemical compounds, either purchased from ChemDiv or synthesized in the laboratory, were suspended in DMSO and titrated as detailed in each figure. The DMSO concentration in the reaction mixture was kept constant at or below 5%. RPA was incubated with inhibitor or DMSO control in reaction buffer for 30 minutes before the addition of DNA. Reactions were incubated for 5 minutes at room temperature and products separated via 6% native polyacrylamide gel electrophoresis. The bound and unbound fractions were then quantified by phosphor-imager analysis using ImageQuant software (Molecular Dynamics, CA) and IC50 values calculated by non-linear regression using SigmPlot (Sysat). For EMSA reactions with RPA DBD-A/B, 150 nM DBD-A/B was used and electrophoresis was performed at 4° C. All other conditions were identical to those described for the full length RPA. RPA was incubated with the above compounds at a concentration range of 1-125 μM |
| Affinity data for this assay | |
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