Detailed specificity analysis of antibodies binding to modified histone tails with peptide arrays

Abstract

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers which are directed towards modified histone tails. The arrays contained 384 peptides from 8 different regions of the N-terminal tails of histones, viz. H3 1-19, 7-26, 16-35 and 26-45, H4 1-19 and 11-30, H2A 1-19 and H2B 1-19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profile. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed towards the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.

Acknowledgements

Thanks are due to Sandra Becker for technical support. This work has been supported in part by the NIH DK08267 grant.

Figures and Tables

Figure 1 (A) Design of the peptide array used in this study (detailed information on the sequence and modification of each peptide is given in Sup. Table 1). Here the image obtained with antibody #1 is used for illustration. (B) Antibody binding to independently synthesized peptides. Spot intensities were averaged from the two internal repeats of the array and compared between arrays that were synthesized independently. One image obtained with H3K4me3 is chosen for illustration. The scatter plots show a comparison of the intensity of peptide binding in both arrays. The r value refers to the Pearson correlation coefficient of both intensities. (C) Reproducibility of peptide binding intensities between different peptides on the same array. For several antibodies, the binding intensities to all peptides containing the primary epitope (after exclusion of false negatives) were averaged and plotted in log scale (orange columns, the error bars display the standard deviations). As background, binding intensities to the 100 weakest spots were used (blue columns, the error bars display the standard deviations). For antibody numbers cf. Supplemental Table 2.

Figure 2 Internal reproducibility of the peptide binding. Spot intensities were quantified on the two internal repeats of the array and compared. One image obtained with H3K4me3 is chosen for illustration. Examples of the results are given for several antibodies. The box plots show the number of pairs of spots within certain error margins obtained after normalization to the maximum binding. The scatter plots show a comparison of the intensity of each peptide in both arrays. The r value refers to the Pearson correlation coefficient of both intensities.

Figure 3 Examples of antibody specificity analysis. The upper part of each panel shows the image of the array. Peptide spots are annotated on the left copy of the duplicates, color coded as described below the image. On the left side, spots are annotated as follows: all peptides containing the primary PTM are shaded in green, false negatives (or very weakly bound peptides which contain the primary PTM) are highlighted with red arrows. False positives are encircled in orange or violet color. The secondary PTMs present in the false negative, and false positive spots are specified below the pictures. In the lower part a scatter plot of the binding intensities to both repeats is shown.

Figure 4 Examples of results obtained with different antibodies binding to H3K27me3. Peptide spots are annotated on the left copy of the duplicates, color coded as described next to the image.

Figure 5 Summary of antibody specificities. In this graph, the specificity factor for binding at the target site (SF_T) is plotted on the y-axis and the ratio of the specificity factors for binding at the target site and the best non-target site (SF_T/SF_N) on the x-axis. Best antibodies are located in the upper right corner of the graph. Some examples of antibodies are shown on the right side, for coloring cf. Supplemental Table 1.

Table 1 Compilation of the results of the specificity analyses with different antibodies