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Binding of the geldanamycin derivative 17 DMAG to Hsp90 measured with fluorescence label and label free

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Hsp90, fluorescently labeled with NT647, was used at a concentration of ~ 40 nM. The 17- DMAG stock was dissolved in 50 % (v/v) ethanol at a concentration of 500 µM. We used 50 µM 17-DMAG as the highest concentration for the serial dilution. At this concentration the final ethanol concentration still was 5 % (v/v). To make sure that no buffer effects are observed the dilution buffer was prepared accordingly making sure that the final ethanol concentration was ~ 5 % (v/v) in all samples. After 1 min incubation at room temperature the samples were loaded into standard treated capillaries. Fig. 2 shows the initial capillary scan, the shape of the MST curves and the resulting binding curve. The initial capillary scan already indicated slight unspecific binding of the labeled Hsp90 to the capillary walls. Subsequent analysis of the MST curves revealed high fluctuations in the data. Fig. 2 Binding of 17-DMAG to Hsp90 in standard treated capillaries: initial capillary scan (upper), shape of the MST curves (inset) and the resulting data points (n = 3 independent measurements, lower). The initial capillary scan already indicated slight unspecific binding of the labeled Hsp90 to the capillary walls as does the high standard deviation of the data points. The presence of double peaks in the capillary scan after the measurement in standard treated capillaries (Fig. 3) clearly indicated unspecific binding of the labeled Hsp90 to the capillary walls. Fig. 3 Capillary scan after the measurement in standard treated capillaries: the double peaks clearly indicate unspecific binding of labeled Hsp90 to the capillary walls. We therefore tested hydrophobic and hydrophilic capillaries. Fig. 4 shows the initial scan, the MST curves and the resulting binding curve for the measurement in hydrophilic capillaries. Fig. 4 Measurement of Hsp90 vs. 17-DMAG in hydrophilic capillaries: initial capillary scan (upper), shape of the MST curves (inset) and the resulting binding curve (n = 3 independent measurements, lower). The calculated K d of 0.503 ± 0.099 µM is in good agreement with the published K d of 0.35 ± 0.04 µM (Onuoha et al. 2007). In comparison to the standard capillaries the standard deviations of the data were small. Also after the measurement no unspecific binding of labeled Hsp90 was observed (Fig.5).

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