Data Sheet PRMT6 Direct Activity Assay Kit
Contents
1. 20 ul Keep diluted enzyme on ice until use Discard any unused diluted enzyme after use 7 Add 20 ul of 1X HMT assay buffer 6 to the wells designated Blank 8 Initiate reaction by adding 20 ul of diluted PRMT6 enzyme to the wells designated Positive Control Substrate Control and Test Sample Incubate at room temperature for 1 hour 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 Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Bioscience 9 Wash the plate three times with 200 ul TBST buffer Blot dry onto clean paper towels 10 Add 100 ul of Blocking buffer to every well Shake on a rotating platform for 10 min Remove supernatant as above Positive Test Substrate Blank Control Sample Control 4x HMT assay buffer 6 7 5 ul 7 5 ul 7 5 ul 7 5 ul 400 uM S adenosylmethionine 2 5 ul 2 5 ul 2 5 ul H2O 15 0 ul 15 0 ul 17 5 ul 15 0 ul Test Inhibitor Activator 5 ul Inhibitor buffer no inhibitor 5 ul 5 ul 5 ul 1X HMT assay buffer 6 20 ul Diluted PRMT6 1 5 ng l 20 ul 20 ul 20 ul Total 50 ul 50 ul 50 ul 50 ul Step 2 1 Dilute Primary antibody 4 100 fold2. 51040 50 ug PRMT1 expressed in Sf9 cells 51041 20 ug PRMT3 expressed in E coli 51043 50 ug PRMT4 expressed in HEK293 51047 20 ug PRMT5 expressed in HEK293 51045 20 ug PRMT5 MEP50 expressed in Sf9 cells 51048 20 ug PRMT6 expressed in HEK293 51046 20 ug PRMT8 expressed in Sf9 cells 51052 20 ug PRMT1 Chemiluminescent Assay Kit 52004L 96 reactions PRMT3 Chemiluminescent Assay Kit 52005L 96 reactions PRMT4 Chemiluminescent Assay Kit 52041L 96 reactions PRMT5 Chemiluminescent Assay Kit 52002 96 reactions PRMT1 Homogeneous Assay Kit 52052 384 reactions PRMT3 Homogeneous Assay Kit 52055 384 reactions PRMT5 Homogeneous Assay Kit 52054 384 reactions PRMT6 Homogeneous Assay Kit 52056 384 reactions PRMT8 Homogeneous Assay Kit 52058 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 bpsbioscience com Please visit our website at www bpsbioscience com 130822 130822 Bioscience TROUBLESHOOTING GUIDE Problem Luminescence signal of positive control reaction is weak Possible Cause PRMT6 enzyme has lost activity 6044 Cornerstone Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Enzyme loses activity upon repeated freeze thaw cycles Use fresh enzyme PRMT6 BPS Bioscience 51049 Store enzyme i
3. microwells by adding 150 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 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 contents of the tube Aliquot S adenosylmethionine into single use aliquots and store at 80 C Note S adenosylmethionine is very sensitive to freeze thaw cycles Avoid multiple freeze thaw cycles 3 Prepare the master mixture N wells x 7 5 ul 4X HMT assay buffer 6 2 5 ul 400 uM S adenosylmethionine 15 ul water Add 25 ul of master mixture to all wells labeled Positive Control Test Sample and Blank For wells labeled Substrate control add 7 5 ul 4X HMT assay buffer 6 17 5 ul water 4 Add 5 ul of inhibitor solution of each well designated Test Inhibitor For the Positive Control Substrate Control and Blank add 5 ul of the same solution without inhibitor inhibitor buffer 5 Thaw PRMT6 enzyme on ice Upon first thaw briefly spin tube containing enzyme to recover full content of the tube Aliquot PRMT6 enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C Note PRMT6 enzyme is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 6 Dilute PRMT6 enzyme in 1X HMT assay buffer 6 at 1 5 ng ul 20 100 ng
4. with Blocking buffer 2 Add 100 ul per well Incubate 1 hour at room temperature with slow shaking 3 Wash plate with TBST buffer and Blocking buffer as in step 1 9 and 1 10 Step 3 1 Dilute Secondary HRP labeled antibody 2 1 000 fold with Blocking buffer Add 100 ul per well Incubate for 30 min at room temperature with slow shaking Wash plate with TBST buffer and Blocking buffer as in step 1 9 and 1 10 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 Blank value is subtracted from all other values 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 130822 6044 Cornerstone Court W Ste E 6 San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com 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 sp
5. 6044 Cornerstone Court W Ste E Bi San Diego CA 92121 Tel 1 858 829 3082 lOSCICNCE Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet PRMT 6 Direct Activity Assay Kit Catalog 52046 Size 96 reactions DESCRIPTION The PRMT6 Direct Activity Assay kit is designed to measure PRMT6 activity for screening and profiling applications The PRMT6 Direct Activity Assay Kit comes in a convenient format with a 96 well plate precoated with histone H4 peptide substrate the antibody against methylated arginine3 residue of Histone H4 the secondary HRP labeled antibody S adenosylmethionine methyltransferase assay buffer and purified PRMT6 enzyme for 96 enzyme reactions The key to the PRMT6 Direct Activity 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 Next primary antibody is added Finally the plate is treated with an HRP labeled secondary antibody followed by addition of the HRP substrate to produce chemiluminescence that can then be measured using a chemiluminescence reader COMPONENTS Catalog Component Amount Storage 51049 PRMT6 20 ug 80 C 52120 400 uM S adenosylmethionine 250 ul 80 C 52150 Primary antibody 4 100 ul 80 C 52131H
6. Secondary HRP labeled antibody 2 10 ul 80 C 4x HMT assay buffer 6 3 ml 20 C Avoid 52100 Blocking buffer 50 ml 4 C freeze HRP chemiluminescent substrate A 6 ml 4 C thaw transparent bottle cycles HRP chemiluminescent substrate B 6 ml 4 C brown bottle 8 well strip plate module precoated 1 plate 4 C with histone substrate 12 x 8 well strips 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 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 130822 130822 6044 Cornerstone Court W Ste E 6 San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com CONTRAINDICATIONS DMSO gt 1 strong acids or bases ionic detergents high salt STABILITY One year from date of receipt when stored as directed REFERENCE 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
7. ectrophotometry To properly read chemiluminescence make sure the plate reader is set for LUMINESCENCE mode Typical integration time is 1 second delay after plate movement is 100 msec Do not use a filter when measuring light emission Typical settings for the Synergy 2 BioTek plate reader are use the hole position on the filter wheel Optics position Top Read type endpoint Sensitivity may be adjusted based on the luminescence of a control assay without enzyme typically we set this value as 100 Example of Assay Results PRMT6 activity 13000 12000 11000 10000 9000 8000 7000 6000 4 5000 4000 n 3000 2000 1000 Luminescence 0 100 200 300 400 500 PRMT6 ng PRMT6 enzyme activity measured using the PRMT6 Chemiluminescent Assay Kit BPS Bioscience 52046 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 130822 6044 Cornerstone Court W Ste E 6 San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com RELATED PRODUCTS PRMT1 expressed in E coli
8. 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
9. n single use aliquots Increase time of enzyme incubation Increase enzyme concentration inhibiting the enzyme Antibody reaction is Increase time for primary antibody insufficient incubation Avoid freeze thaw cycles of antibodies Incorrect settings on Record light signals at 5 second instruments intervals Refer to instrument instructions for 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 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 PRMT6 BPS Bioscience 51049 to create a standard curve OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To p
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