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. Author manuscript; available in PMC: 2008 Dec 15.
Published in final edited form as: Anal Chem. 2007 Nov 13;79(24):9398–9402. doi: 10.1021/ac701701k

Figure 4.

Figure 4

Comparison of specific and non-specific binding of LG100268 and 9-cis-RA to recombinant human RXRα during ultrafiltration LC-MS-MS screening using the one-filter and two-filter procedures. In the MRM chromatograms, the solid lines represent incubations with active RXRα, and the dashed lines represent the use of denatured RXRα as a control for non-specific binding. The concentrations of ligand and protein were each 1 μM, and each incubation was carried out for 2 h. A) When the RXRα-ligand complexes were washed with ammonium acetate to remove unbound compounds and then dissociated using methanol/water (90:10; v/v) using the same ultrafiltration filter, non-specific binding to the membrane produced a strong signal for LG100268 and 9-cis-RA. B) Transferring the RXRα-ligand complexes after the washing step to a clean ultrafiltration filter for dissociation resulted in much less non-specific binding.