Application Notes

Binding of histone peptides to chromatin assembly factor I (CAF-I) p48 subunit

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3 Fig. 5 Binding of NT647-labeled H3NL peptide to p48 in hydrophilic treated capillaries: shape of the MST-curves and the resulting binding curve. The binding curve represents the data points from 3 measurements. The calculated K d is 4.2 ± 0.93 µM. This value is in good agreement with the K d determined for the H3NL – p48 interaction when p48 was labeled, which gave a dissociation constant of 6.95 µM (data not shown). Conclusion The study provides an example for measurements of protein-peptide interactions with MicroScale Thermophoresis. In this study we worked with labeled protein as well as labeled peptide. It also illustrates the high content information of the measurement which allows to directly adjust and optimize the assay conditions either by changing the type of capillaries or by adjusting the buffer conditions. Material and Methods Assay conditions For the experiments either p48 or the H3NL peptide were labeled with the Monolith NT™ Protein Labeling Kit RED (Cat#L001) according to the supplied labeling protocol. Labeled protein / peptide was used at a concentration of ~ 50 nM. H4NL peptide was titrated in 1:1 dilutions beginning at 250 µM, H3NS peptide was titrated in 1:1 dilutions beginning at 883 µM, and p48 was titrated in 1:1 dilutions beginning at 15 µM. The p48-NT-647 labeled – H3NS and the p48- NT-647 labeled – H3NL (data not shown) experiments were performed in HEPES Buffer with 100 mM NaCl and measured in standard treated capillaries (Cat#K002) and hydrophilic treated capillaries (Cat#K004), respectively. Both the p48-NT-647 labeled – H4NL experiment and the H3NL-NT647 labeled – p48 experiment were performed in MST optimized buffer (50 mM Tris buffer pH 7.4 containing 150 mM NaCl, 10 mM MgCl 2 and 0.05 % Tween-20) and measured in hydrophilic capillaries (Cat#K004). For the H3NL- NT-647 labeled – p48 experiment the MST optimized buffer was supplemented with glycerol (final concentration 5 %) to ensure the same buffer condition for all dilutions of p48. All binding reactions were incubated for 10 min at room temperature before they were loaded into the capillaries. Instrumentation The measurements were done on a NanoTemper Monolith NT.015 instrument. All measurements were performed at 40 % LED and 40 % MST power, Laser-On time was 30 sec, Laser-Off time 5 sec. References Davey et al., Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution. J.Mol.Biol. 2002 319: 1097-111 Verreault et al., Nucleosome Assembly by a Complex of CAF- 1 and Acetylated Histones H3/H4 Cell 1996, Volume 87, Issue 1: 95-104 © 2011 NanoTemper Technologies GmbH

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