Data Sheet - BPSBioscience.com
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1. the plate onto clean paper towels to remove liquid 2 Thaw JMJD2C on ice Upon first thaw briefly spin tube containing enzyme to recover full content of the tube Aliquot JMJD2C enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C Note JMJD2C is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 3 Dilute JMJD2C in 1X JMJD2C assay buffer at 10 ng ul Keep diluted enzyme on ice until use Discard any unused diluted enzyme after use 4 Using master mixes as much as possible add the following reagents to the microwells in duplicate Positive Test Test Blank Control Sample Inhibitor JMJD2C 10 ng ul 20 ul 20 ul JMJD2C containing sample 20 ul 4x JMJD2C assay buffer 1 12 5 ul 12 5 ul 12 5 ul 12 5 ul Test Inhibitor Activator X ul H2O 17 5 ul 17 5 ul 17 5 X ul 37 5 ul Total 50 ul 50 ul 50 ul 50 ul 5 Add the entire reaction mixture 50 ul to the substrate coated black plate OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE Incubate at room temperature for 1 hour Wash the plate three times with TBST buffer Blot dry onto clean paper towels Add 100 ul of Blocking buffer to every well Shake on a rotating platform for 10 min Remove supernatant as above 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 bpsbioscienc2. 120919 6044 Cornerstone Court W Ste E Bi E San Diego CA 92121 Tel 1 858 829 3082 lOSCICNCE Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet JMJD2C Chemiluminescent Assay Kit Catalog 50405 DESCRIPTION The JMJD2C Chemiluminescent Assay Kit is designed to measure JMJD2C activity for screening and profiling applications JMJD2C is a JmjC domain protein that exhibits demethylation activity toward H3 K9Me3 and H3 K36Me3 The JMJD2C Chemiluminescent Assay Kit comes in a convenient format with a 96 well plate precoated with the methylated histone H3 peptide substrate primary antibody the secondary HRP labeled antibody demethylase assay buffer and purified JMJD2C for 100 enzyme reactions The key to the JMJD2C Chemiluminescent 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 JMJD2C enzyme is incubated with a sample containing assay buffer for one hour Next primary antibody is added Finally the plate is treated with an HRP labeled secondary antibody followed by the addition of the HRP substrate to produce chemiluminescence that can then be measured using a chemiluminescence reader COMPONENTS Catalog Component Amount Storage 50105 JMJD2C GASC1 KDM4C 20 ug 80 C 52140E Primary antibody 5 25 ul 80 C 52130H Secondary HRP labele
3. C 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
4. aw cycles Use fresh same as blank value JMJD2C BPS Bioscience 50105 Store enzyme in single use aliquots Increase time of enzyme incubation Increase enzyme concentration Antibody reaction is Increase time for primary antibody insufficient incubation Avoid freeze thaw cycles of antibodies Incorrect settings on Refer to instrument instructions for instruments settings to increase sensitivity of light detection Chemiluminescent Chemiluminescent solution should be reagents mixed too used within 15 minutes of mixing soon Ensure both reagents are properly mixed Luminescent signal is Inaccurate Run duplicates of all reactions erratic or varies widely pipetting technique Use a multichannel pipettor among wells Use master mixes to minimize errors Bubbles in wells Pipette slowly to avoid bubble formation Tap plate lightly to disperse bubbles be careful not to splash between wells Background signal to noise Insufficient washes Increase number of washes ratio is high Increase wash volume Increase Tween 20 concentration to 0 1 in TBST Sample solvent is Run negative control assay including inhibiting the enzyme solvent Maintain DMSO level at lt 1 Increase time of enzyme incubation Results are outside the linear assay range of the Use different concentrations of JMJD2C BPS Bioscience 50105 to create a standard curve OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTI
5. d antibody 1 10 ul 80 C Avoid 52304 4x JMJD2C assay buffer 3 ml 20 C ireazel 52100 Blocking buffer 50 ml 4 C thaw HRP chemiluminescent substrate 6 ml each 4 C cycles ycles 2 components Black microplate precoated with 1 4 C histone substrate MATERIALS REQUIRED BUT NOT SUPPLIED TBST buffer 1 x TBS pH 8 0 containing 0 05 Tween20 Luminometer or fluorescent microplate reader capable of reading chemiluminescence Adjustable micropipettor and sterile tips Rotating or rocker platform APPLICATIONS Great for studying enzyme kinetics and HTS applications CONTRAINDICATIONS DMSO gt 1 strong acids or bases ionic detergents high salt 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 120919 6044 Cornerstone Court W Ste E n 7 San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com STABILITY One year from date of receipt when stored as directed REFERENCE Whetstine JR ea Cell 2006 125 467 ASSAY PROTOCOL All samples and controls should be tested in duplicate Step 1 1 Rehydrate the microwells by adding 200 ul of TBST buffer 1 x TBS pH 8 0 containing 0 05 Tween 20 to every well Incubate 15 minutes at room temperature Tap
6. e com Please visit our website at www bpsbioscience com 6044 Cornerstone Court W Ste E f San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Step 2 1 Dilute Primary antibody 5 400 fold with Blocking buffer 2 Add 100 ul per well Incubate 1 hour at room temperature with slow shaking 3 Wash plate three times with TBST buffer and incubate in Blocking buffer as in steps 1 6 and 1 7 Step 3 1 Dilute Secondary HRP labeled antibody 1 1 000 fold with Blocking buffer Add 100 ul per well Incubate for 30 min at room temperature with slow shaking 3 Wash plate with TBST buffer and incubate in Blocking buffer as in step 1 6 and 1 7 Just before use mix on ice 50 ul HRP chemiluminescent substrate A and 50 ul HRP chemiluminescent substrate B and add 100 ul per well Discard any unused chemiluminescent reagent after use 5 Immediately read sample in a luminometer or microtiter plate capable of reading chemiluminescence Example of Assay Results JMJD 2C activity 1200 1000 800 600 Luminescence 400 200 0 100 200 300 400 500 JMJD 2C ng JMJD2C enzyme activity measured using the JMJD2C Chemiluminescent Assay Kit BPS Bioscience 50405 Data shown is lot specific For lot specific information please contact BPS Bioscience Inc at info bpsbioscience com OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To p
7. lace 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 120919 6044 Cornerstone Court W Ste E f San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com RELATED PRODUCTS Product Name Catalog Size JMJD2A recombinant protein 50103 20 ug JMJD2B recombinant protein 50104 20 ug JMJD2C recombinant protein 50105 20 ug JMJD3 recombinant protein 50115 20 ug JMJD2A Homogeneous Assay Kit 50413 384 reactions JMJD2B Homogeneous Assay Kit 50414 384 reactions JMJD2C Homogeneous Assay Kit 50415 384 reactions LSD1 Assay Kit 96 well 50106 100 reactions LSD1 recombinant protein 50100 50 ug LSD1 substrate 50101 500 ul COREST recombinant protein 50274 100 yg 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 120919 120919 Bioscience TROUBLESHOOTING GUIDE 6044 Cornerstone Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Problem Possible Cause Luminescence signal of JMJD2C has lost Enzyme loses activity upon repeated positive control reaction is activity freeze th
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