Objective

Results from sequencing large numbers of normal vs. tumor samples from the cancer genome atlas project (TCGA) revealed a great number of potential new therapeutic targets among epigenetic enzymes and factors. An outcome of this large scale project is that there is significant interest in developing screening strategies for many of the classes of these proteins, including histone deacetylases (HDACs), histone acetyltransferases (HATs), lysine methyltransferases (KMTs), lysine demethylases (KDMs), and bromodomain containing proteins (bromodomains). While the activity of some of these targets have yet to be validated using screening platforms that use peptide substrates (i.e. Perkin Elmer AlphaLISA), Active Motif has produced a protein toolbox of reagents including active enzymes, recombinant substrates, and detection antibodies for homogenous assay platforms such as AlphaLISA. homogenous assay platforms such as AlphaLISA. We have validated reagents used in these assays for targets HDAC3, LSD1, p300, SETDB1, and BRD family members to ensure our ability to validate these proteins for performance in screening assays Active Motif has produced a protein toolbox of reagents including active enzymes, recombinant substrates, and detection antibodies for homogenous assay platforms such as AlphaLISA. We have therefore validated reagents used in these assays for targets HDAC3, LSD1, p300, SETDB1, and BRD family members to ensure our ability to validate these proteins for performance in screening assays. In general, IC50 values of these proteins, as assessed by AlphaLISA assays, are well within published results for the various compounds that were used in the evaluation. This protein toolbox continues to expand to include “designer” oligo nucleosomes, recombinant nucleosomes which include site directed installation of modified amino acids. Additional candidates can now be screened using these alternate nucleosome based substrates.

Methods

homogenous assay platforms such as AlphaLISA.

Results

We have validated reagents used in these assays for targets HDAC3, LSD1, p300, SETDB1, and BRD family members to ensure our ability to validate these proteins for performance in screening assays

Conclusion

Active Motif has produced a protein toolbox of reagents including active enzymes, recombinant substrates, and detection antibodies for homogenous assay platforms such as AlphaLISA. We have therefore validated reagents used in these assays for targets HDAC3, LSD1, p300, SETDB1, and BRD family members to ensure our ability to validate these proteins for performance in screening assays. In general, IC50 values of these proteins, as assessed by AlphaLISA assays, are well within published results for the various compounds that were used in the evaluation. This protein toolbox continues to expand to include “designer” oligo nucleosomes, recombinant nucleosomes which include site directed installation of modified amino acids. Additional candidates can now be screened using these alternate nucleosome based substrates.