JMJD2E Homogeneous Assay Kit
Contents
1. MATERIALS REQUIRED BUT NOT SUPPLIED AlphaLlSA anti mlgG acceptor beads 5 mg ml PerkinElmer AL105C AlphaScreen Streptavidin conjugated donor beads 5 mg ml PerkinElmer 6760002S Optiplate 384 PerkinElmer 6007290 AlphaScreen microplate reader Adjustable micropipettor and sterile tips APPLICATIONS Great for studying enzyme kinetics and HTS applications CONTRAINDICATIONS Green and blue dyes that absorb light in the AlphaScreen signal emission range 520 620 nm such as Trypan Blue Avoid the use of the potent singlet oxygen quenchers such as sodium azide NaNs3 or metal ions Fe Fe Cu Zn and Ni The presence of culture medium RPMI 1640 at gt 1 leads to signal reduction due to the presence of excess biotin and iron in this medium MEM which lacks these components does not affect AlohaScreen assays STABILITY At least one year from date of receipt when stored as directed OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 6044 Cornerstone Ct West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com ASSAY PROTOCOL All samples and controls should be tested in duplicate Step 1 1 Thaw JMJD2E on ice Upon first thaw briefly2. 121113F 6044 Cornerstone Ct West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet JMJD2E Homogeneous Assay Kit Catalog 50417 DESCRIPTION The JMJD2E Homogeneous Assay Kit is designed to measure activity of JMJD2E for screening and profiling applications JMJD2E also known as KDMA4DL lysine K specific demethylase 4D like is a JmjC domain protein that exhibits demethylation activity toward H3 K9Me3 and H3 K36Me3 The JMJD2E Homogeneous Assay Kit comes in a convenient AlphaLISA format with biotinylated histone H3 peptide substrate primary antibody demethylase assay buffer and purified JMJD2E for 384 enzyme reactions The key to the JMJD2E Homogeneous Assay Kit is a highly specific antibody that recognizes demethylated substrate With this kit only three simple steps on a microtiter plate are required for methyltransferase detection First a sample containing JMJD2E enzyme is incubated with the biotinylated substrate for two hours Next acceptor beads and primary antibody are added then donor beads followed by reading the Alpha counts COMPONENTS Cat Component Amount Storage 50118 JMJD2E 40 ug 80 C 52140E Primary antibody 5 40 ul 80 C Avoid Biotinylated histone H3 400 ul 80 C freeze thaw peptide substrate cycles 52305 4x JMJD2E assay buffer 2 ml 20 C 4x Detection buffer 2 ml 20 C
3. TIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com
4. om temperature 2 Read Alpha counts OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 121113F 121113F 6044 Cornerstone Ct West Ste E San Diego CA 92121 amp Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Example of Assay Results JMJD2E KDM4DL activity 15000 12500 10000 7500 A counts 5000 2500 10 20 30 40 50 60 70 80 90 100 110 JMJD2E KDMAL ng JMJD2E enzyme activity measured using the JMJD2E Homogeneous Assay Kit BPS Bioscience 50417 Data shown is lot specific For lot specific information please contact BPS Bioscience Inc at info bpsbioscience com REFERENCE Whetstine J R et al Cell 2006 125 467 RELATED PRODUCTS JMJD2A recombinant protein 50103 20 ug JMJD2B recombinant protein 50104 20 ug JMJD2C recombinant protein 50105 20 ug JMJD2E KDM4DL recombinant protein 50118 20 ug JMJD3 recombinant protein 50115 20 ug JMJD6 recombinant protein 50140 20 ug JMJD2A Homogeneous Assay Kit 50413 384 rxns JMJD2B Homogeneous Assay Kit 50414 384 rxns JMJD2C Homogeneous Assay Kit 50415 384 rxns AlphaScreen and AlphaLISA are registered trademarks of PerkinElmer Inc OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEU
5. spin tube containing enzyme to recover full content of the tube Aliquot JMJD2E enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C Note JMJD2E is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 2 Dilute JMJD2E in 1X JMJD2E assay buffer at 40 ng ul 100 ng 2 5 ul Keep diluted enzyme on ice until use Discard any unused diluted enzyme after use 3 Using master mixes as much as possible add the following reagents to the microwells in duplicate Reagent Positive Test Sample Blank Control JMJD2E 40 ng ul 2 5 ul 2 5 ul 4x JMJD2E assay buffer 1 2 5 ul 2 5 ul 2 5 ul Biotinylated substrate 1 ul 1 ul 1 ul Test Inhibitor Activator X ul H2O 4 ul 4 Xul 6 5 ul Total 10 ul 10 wl 10 wl 4 Add the entire reaction mixture 10 ul to the well of a 384 well white plate Incubate at room temperature for 1 hour Step 2 Note Protect your samples from direct exposure to light 1 Dilute anti Mouse Acceptor beads PerkinElmer AL105C 1 250 with 1x Detection buffer Add 5 ul per well Shake plate briefly 2 Dilute Primary antibody 5 50 fold with 1x Detection buffer Add 5 ul per well Shake on a rotator platform for 30 minutes at room temperature Step 3 1 Dilute Streptavidin conjugated donor beads PE 6760002S 125 fold with 1x Detection buffer Add 10 ul per well Shake on a rotator platform for 15 minutes at ro
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