Check the specificity of modified Histone antibodies
A lot of antibodies that have been raised against modified Histones, e.g. methylated or acetylated histones, do not seem to have been tested for their specificity. Antibodies against mono-methylated lysines should, for instance, not recognize di- or tri-methylated sites. Or totally different sites which might be methylated as well. Eglehofer et al. reported about the specificity of > 200 antibodies against 57 different Histone modifications and found that more than 25% failed in specificity tests (Nat Struct Mol Biol 18.1, 91-3 ). In their paper they advised rigorous testing and provided an Antibody Validation Database.
Nowadays, and in order to avoid numerous Western blot or Dot blot analysis to seek for Histone-modified antibody specificity, the array technology has been chosen as an attractive approach by numerous laboratories. Such arrays render the validation of the antibody specificity much easier and rapid with only one experiment.
EpiTitan™ Histone Peptide Arrays already represent the next generation in Histone peptide arrays, and are the gold standard against which all other histone peptide arrays are judged.
These arrays feature hundreds of highly purified and well-characterized modified Histone peptides (acetylated, mono-, di- and trimethylated, phosphorylated with a huge number of combinations) spotted onto the substrate.
EpiTitan™ Histone Peptide Arrays are a dramatic improvement over arrays in which the peptides are synthesized on the substrate (SPOT synthesis), a technique that leads to peptides on the array that are of unknown quantity and quality. All the peptides on the EpiTitan™ Histone Peptide Arrays are purified by HPLC and validated by mass spectrometry prior to spotting.
EpiTitan™ Histone Peptide Array features
EpiTitan™ Histone Peptide Arrays are a brilliant tool for antibody users to make sure that they detect the correct (and only the correct) target as well as for antibody producing companies which can use the arrays as quality controls.
- Biotinylated peptides are spotted 12 times each per array on a surface covered with streptavidin to catch the peptides.
- Each array slide comes with two subarrays, thus two samples per slide can be evaluated.
- The protein to be investigated is added and binds to its specific target sequence and modification pattern.
- The detection of binding either starts with an antibody against the protein of interest or an anti tag antibody, should the protein carry the respective tag, and can be visualized using fluorescence scanning or standard ECL techniques – by the way the EpiTitan™ Histone Peptide Array is the only array available on the market which supports both detection methods.
- Protocols for qualitative as well as quantitative analysis of the data are provided.
Interested in more information or using EpiTitan™ Histone Peptide Arrays yourself?
Just leave a comment if you’d like to check the list of histone peptides and the user manual. I’ll be happy to get back to you to tell you more about these Histone arrays and companion reagents (Epigenetic readers and erasers or bioactive small molecules…).