PHF8 (KDM7B) Homogeneous Assay Kit
The PHF8 Homogeneous Assay Kit is designed to measure PHF8activity for screening and profiling applications. PHF8, also known as JHDM1F and KDM7B, is aJumonjiC (JmjC) domain containing histone lysine demethylase that exhibits demethylationactivity toward H3-K9Me2. The PHF8 Homogeneous Assay Kit comes in a convenientAlphaLISA® format (Scheme 1), with biotinylated histone H3 peptide substrate, primaryantibody, demethylase assay buffer, and purified PHF8 for 96 enzyme reactions. The key to thePHF8 Homogeneous Assay Kit is a highly specific antibody that recognizes demethylatedsubstrate. With this kit, only three simple steps on a microtiter plate are required formethyltransferase detection. First, a sample containing PHF8 enzyme is incubated with thebiotinylated substrate. Next, acceptor beads and primary antibody are added, then donor beads,followed by reading the Alpha-counts.
Scheme 1: Our histone demethylase assays utilize highly specific antibodies that recognize demethylated products. First, a sample containing the enzyme is incubated with a biotinylated substrate. Next, acceptor beads and primary antibody are added, then donor beads, followed by reading the Alpha-counts, as shown above.
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AlphaLISA® anti-RIgG acceptor beads, 5 mg/ml (PerkinElmer #AL104C)
AlphaScreen® Streptavidin-conjugated donor beads, 5 mg/ml (PerkinElmer #6760002S)
Optiplate -96 (PerkinElmer #6005290)
AlphaScreen® microplate reader
Adjustable micropipettor and sterile tips
96 Reactions:
Catalog # | Component | Amount | Storage | |
50131 | PHF8 (KDM7B) | 20 μg | -80°C | Avoid freeze/ thaw cycles! |
52140Q4 | Primary antibody 17-4 | 5 μl | -80°C | |
Biotinylated histone H3 peptide substrate | 300 rxns | -80°C | ||
52408 | 4x HDM Assay Buffer 3 | 3 x 1 ml | -80°C | |
52031 | 4x Detection buffer | 2 ml | -20°C |
384 Reactions:
Catalog # | Component | Amount | Storage | |
50131 | PHF8 (KDM7B) | 60 μg | -80°C | Avoid freeze/ thaw cycles! |
52140Q4 | Primary antibody 17-4 | 20 μl | -80°C | |
79845 | Biotinylated histone H3 peptide substrate | 500 rxns | -80°C | |
52408 | 4x HDM Assay Buffer 3 | 3 ml | -80°C | |
52031 | 4x Detection buffer | 2 ml | -20°C |
Horton, J.R., et al. Nature Structural & Molecular Biology 2010; 17(1): 38-43.