Data Sheet Histone H4(R3) Universal
Contents
1. HRP substrate to produce chemiluminescence that can then be measured using a chemiluminescence reader COMPONENTS Cat Components Amount Storage 51040 PRMT1 human recombinant enzyme 500 ng 80 C 51043 PRMT3 human recombinant enzyme 2 ug 80 C 51045 PRMT5 human recombinant enzyme 7 5 Ug 80 C 20 uM S adenosylmethionine 250 ul 80 C Avoid 52150 Primary antibody 4 100 ul 80 C freeze 52131H Secondary HRP labeled antibody 2 10ul 80 C thaw 52170 4x HMT Assay Buffer 2 3 ml 20 C cycles 52100 Blocking buffer 35 ml 4 C HRP chemiluminescent substrate 2 6 ml 4 C components each 96 well plate precoated with histone substrate 1 plate 4 C APPLICATIONS Great for studying enzyme kinetics and HTS applications MATERIALS OR INSTRUMENTS 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 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 140714 6044 Cornerstone Court W Ste E Bi San Diego CA 92121 Tel 1 858 829 3082 lOSCIEMNCEe Fax 1 858 481 8694 Email info bpsbioscienc2. bpsbioscience com Please visit our website at www bpsbioscience com 140714 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 Solution Luminescence weak signal of positive control reaction is Methyltransferase enzyme has lost activity Enzyme loses activity upon repeated freeze thaw cycles Use fresh enzymes PRMT1 BPS Bioscience 51040 PRMT3 51043 PRMT5 51045 Store enzymes 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 See section on Chemiluminescence above Reading Chemiluminescent reagents mixed too soon Chemiluminescent solution should be used within 15 minutes of mixing Ensure both reagents are properly mixed Chemiluminescence signal is erratic or varies widely Inaccurate pipetting technique Run duplicates of all reactions Use a multichannel pipettor 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
3. 0 ul 9 Initiate reaction by adding 20 ul of diluted enzyme to the wells designated Positive Control Substrate Control and Test Inhibitor Incubate at room temperature for 1 hour 10 Remove the supernatant from the wells and wash three times with 200 ul TBST buffer Blot dry onto clean paper towels 11 Add 100 ul of Blocking Buffer to every well Shake on a rotating platform for 10 min Remove supernatant as above Step 2 1 Dilute Primary antibody 4 100 fold with Blocking buffer 2 Add 100 ul per well Incubate 1 hour at room temperature with slow shaking 3 Remove the supernatant from the wells and wash the strip three times with 200 ul TBST buffer and incubate in Blocking Buffer as described in steps 1 10 and 1 11 Step 3 1 Dilute Secondary HRP labeled antibody 2 1 000 fold with Blocking buffer 2 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 steps 1 10 and 1 11 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 140714 6044 Cornerstone Court W Ste E E San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com 4 Just before us
4. 6044 Cornerstone Court W Ste E Bi San Diego CA 92121 Tel 1 858 829 3082 lOSCICENCE Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet Histone H4 R3 Universal Methyltransferase Assay Kit Catalog 52074 DESCRIPTION The Histone H4 R3 Universal Methyltransferase Assay Kit is designed for the detection of histone H4 R3 methyltransferase HMT activity using purified enzymes or cell extracts Histone H4 R3 methyltransferases are enzymes that catalyze the transfer of a methyl group from the cofactor S adenosylmethionine to arginine 3 residue of histone H4 The Histone H4 R3 Universal Methyltransferase Assay Kit comes in a convenient format with a 96 well plate precoated with histone H4 peptide substrate the antibody against methylated arginine residue of Histone H4 the secondary HRP labeled antibody S adenosylmethionine methyltransferase assay buffer and purified PRMT1 PRMT3 and PRMT5 enzymes for 100 enzyme reactions The key to the Histone H4 R3 Universal Methyltransferase Assay Kit is a highly specific antibody that recognizes methylated R3 residue of Histone H4 With this kit only three simple steps on a microtiter plate are required for methyltransferase detection First S adenosylmethionine is incubated with a sample containing assay buffer and methyltransferase enzyme for one hour Next primary antibody is added Finally the plates are treated with an HRP labeled secondary antibody followed by addition of the
5. e 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 Blank value is subtracted from all readings Reading Chemiluminescence Chemiluminescence is the emission of light luminescence which results from a chemical reaction The detection of chemiluminescence requires no wavenlength selection because the method used is emission photometry and is not emission spectrophotometry To properly read chemiluminescence make sure you are using your plate reader in a LUMINESCENCE mode Typical integration time is 1 second delay after plate movement is 100 msec Make sure you don t have filter when emit the light Synergy 2 BioTek use hole position on filter wheel Optics position Top Read type endpoint Sensitivity may be adjusted based on luminescence of a control without enzyme typically we set this value as 100 when using Synergy 2 plate reader Example of Assay Results PRMT5 activity PRMT1 activity PRITS activity m 12000 y 200 a 3 S Luminescence en s 8 Luminescence Luminescence a s 0 1 2 3 4 5 0 0 05 10 15 20 0 20 40 60 80 100 Enzyme ng Enzyme ng Enzyme ng PRMT1 PRMT3 and PRMT5 enzyme activity measured using the Histone H4 R3 Methyltransferase Assay Kit BPS Bio
6. e com CONTRAINDICATIONS DMSO gt 1 strong acids or bases ionic detergents high salt REFERENCE S Dillon SC Zhang X Trievel RC Cheng X Genome Biology 2005 6 227 ASSAY PROTOCOL All samples and controls should be tested in duplicate Step 1 1 Rehydrate the microwells by adding 150 ul of TBST buffer 1x TBS pH 8 0 containing 0 05 Tween 20 to every well Incubate 15 minutes at room temperature Tap the plate onto clean paper towels to remove liquid 2 Thaw S adenosylmethionine on ice Upon first thaw briefly spin tube containing S adenosylmethionine to recover full content of the tube Aliquot S adenosylmethionine into single use aliquots and store at 80 C Note S adenosylmethionine very sensitive to freeze thaw cycles Avoid multiple freeze thaw cycles 3 Prepare master mix N wells x 7 5 ul 4x HMT Assay Buffer 2 2 5 ul S adenosylmethionine 15 ul H2O Add 25 ul of master mixture to all wells labeled Positive Control Test Inhibitor and Blank For wells labeled Substrate Control add 7 5 uL 4x HMT Assay Buffer 2 17 5 ul H20 4 Add 5 ul of inhibitor solution to each well designated Test Inhibitor 5 For the Positive Control Substrate Control and Blank add 5 ul of the same solution without inhibitor inhibitor buffer 6 Thaw PRMT1 PRMT3 and PRMT5 enzymes on ice Upon first thaw briefly spin tube containing enzyme to recover full content of the
7. science 52074 Luminescence was measured using a Bio Tek fluorescent microplate reader 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 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 140714 6044 Cornerstone Court W Ste E E San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com RELATED PRODUCTS PRMT1 recombinant protein E coli 51040 50 ug PRMT1 recombinant protein Sf9 51041 20 ug PRMT83 recombinant protein 51043 50 ug PRMT4 CARM 1 recombinant protein 51047 20 ug PRMT5 recombinant protein HEK293 51045 20 ug PRMT5 MEP50 recombinant protein Sf9 51048 20 ug PRMT6 recombinant protein 51046 20 ug PRMT1 Chemiluminescent Assay Kit 52004L 100 reactions PRMT3 Chemiluminescent Assay Kit 52005L 100 reactions PRMT5 Chemiluminescent Assay Kit 52002L 100 reactions PRMT1 Homogeneous Assay Kit 52054 384 reactions PRMT3 Homogeneous Assay Kit 52055 384 reactions PRMT5 Homogeneous Assay Kit 52052 384 reactions 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
8. tube Aliquot PRMT1 PRMT3 and PRMT5 enzymes into single use aliquots Store remaining undiluted enzymes in aliquots at 80 C Note All 3 enzymes are very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzymes 7 Dilute PRMT1 PRMT3 and PRMT5 in 1x HMT Assay Buffer 2 to 0 1 0 5 ng ul 2 10 ng 20 ul 1 2 5 ng ul 20 50 ng 20 ul and 5 10 ng ul 100 200 ng 20 ul respectively Keep diluted enzymes on ice until use Discard any unused diluted enzyme after use 8 Add 20 ul of 1x HMT Assay Buffer 2 to the well designated Blank 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 140714 Bioscience 6044 Cornerstone Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Positive Test Substrate Blank Control Sample Control 4x HMT assay buffer 2 7 5 ul 7 5 ul 7 5 ul 7 5 ul 20 uM S adenosylmethionine 2 5 ul 2 5 ul 2 5 ul H2O 15 ul 15 ul 17 5 ul 15 ul Test Inhibitor Activator 5 ul Inhibitor buffer no inhibitor 5 ul 5 ul 5 ul 1x HMT assay buffer 2 20 ul PRMT1 0 1 0 5 ng ul or PRMT3 1 2 5 ng ul or PRMT5 5 10 ng l aay eop AM J Total 50 ul 50 ul 50 ul 5
9. wells Background ratio is high signal noise Insufficient washes Be sure to include blocking steps after wash steps Increase number of washes Increase wash volume Increase Tween 20 concentration to 0 1 in TBST Sample solvent is inhibiting the enzyme Run negative control assay including solvent Maintain DMSO level at lt 1 Increase time of enzyme incubation Results are outside the linear range of the assay Use different concentrations of enzyme PRMT1 BPS Bioscience 51040 PRMT3 51043 PRMT5 51045 to create a standard curve 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 140714
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