1.1. Validation of Commercial Antibodies Using Peptide Arrays
Validation of commercial antibodies targeting histone modifications is one of the applications where high density peptide arrays offer an advantage, as the target peptide sequences can be systematically explored. This is of particular importance in systems where target sequences are similar or where many isoforms of a single protein exist. In addition, introduction of adjacent PTMs near a central epitope can help clarify the selectivity and specificity of these biology reagents, particularly before using them in long end expensive experiments such as chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). For example, while an antibody targeting serine 10 phosphorylation on histone H3 (H3pS10) exhibited high specificity for this mark, an H3pT11 antibody turned out to be non-selective, recognizing several modifications on the entire H3 N-terminal portion, including pT32, as well as on H2B peptides carrying several different PTMs [10].
CelluSpot™ arrays have also been used to validate 36 commercial antibodies from various sources aiming to determine their specificity towards histone PTMs. Human histone peptides spanning 20-amino-acid N-terminal fragments of each one of the core histones (H2A, H2B, H3 and H4) carrying combinations of 59 PTMs in a 384-well format using internal repeats, were systematically incubated with antibody for 1 h at room temperature. This study yielded robust and reproducible results, with the obtained binding profiles closely matched even when the synthetic origin of the peptides was different. However, despite the fact that most antibodies bound well to the PTM they had been raised for, several failed, suggesting that SPOT techniques are an excellent tool to validate antibodies before using them in complicated biological experiments [15].