Data Sheet PRMT1 Chemiluminescent Assay Kit
Contents
1. Shake on a rotating platform for 10 minutes Remove supernatant as described 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 minutes 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 4 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 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 140623 6044 Cornerstone Court West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com 5 Immediately read sample in a luminometer or microtiter plate reader capable of reading chemilumin2. inhibitors 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 140623 6044 Cornerstone Court West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com 6 Thaw PRMT1 enzyme on ice Upon first thaw briefly spin tube containing enzyme to recover full contents of the tube Aliquot PRMT1 enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C Note PRMT1 enzyme is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 7 Dilute PRMT1 enzyme in 1X HMT assay buffer 2 to 0 1 0 5 ng ul 2 10 ng 20 ul Keep diluted enzyme on ice until use Discard any unused diluted enzyme after use Note optimal enzyme concentration may vary with the specific activity of the enzyme 8 Add 20 ul of 1X HMT assay buffer 2 to the wells designated Blank 9 Initiate reaction by adding 20 ul of diluted PRMT1 enzyme to the wells designated Positive Control Substrate Control and Test Sample Incubate at room temperature for 20 minutes 10 Remove the supernatant from the wells and wash the strip three times with 200 ul TBST buffer Blot dry onto clean paper towels 11 Add 100 ul of Blocking buffer to every well
3. ul 80 C 52131H Secondary HRP labeled antibody 2 10 ul 80 C 52170 4x HMT assay buffer 2 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 96 well plate precoated with histone 1 plate 4 C substrate 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 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 140623 6044 Cornerstone Court West Ste San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Bioscience 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 m
4. 140623 6044 Cornerstone Court West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet PRMT1 Chemiluminescent Assay Kit Catalog 52004L Size 96 reactions DESCRIPTION The PRMT1 Chemiluminescent Assay kit is designed to measure PRMT1 activity for screening and profiling applications The PRMT1 Chemiluminescent Assay Kit comes in a convenient format with wells 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 enzyme for 96 enzyme reactions The key to the PRMT1 Chemiluminescent Assay kit is a highly specific antibody that recognizes methylated R3 residue of Histone H4 With this kit only three simple steps 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 strip plates are 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 51040 PRMT1 human recombinant enzyme 10 ug 80 C 52120 _ 20 uM S adenosylmethionine 250 ul 80 C 52150 Primary antibody 4 100
5. DE San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Problem Possible Cause Solution Luminescence signal of positive control reaction is weak PRMT1 enzyme has Enzyme loses activity upon repeated lost activity freeze thaw cycles Use fresh enzyme PRMT1 BPS Bioscience 51040 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 See section on Reading Chemiluminescence above 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 strip lightly to disperse bubbles be careful not to splash between wells Background signal to noise Insufficient washes Be sure to include blocking steps after ratio is high wash steps Increase number of washes Increase wash volume Increase Tween 20 concentra
6. escence Blank value is subtracted from all other values 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 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 30000 20000 10000 Luminescence 0 0 0 2 5 5 0 7 6 10 0 12 5 PRMT1 ng PRMT1 enzyme activity measured using the PRMT1 Chemiluminescent Assay Kit BPS Bioscience 52004L 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 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 visi
7. icrowells 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 strip 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 2 2 5 ul 20 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 2 17 5 ul water Blank Substrate Positive Test Control Control Sample 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 17 5 ul 15 ul 15 ul Test Inhibitor 5 ul Inhibitor buffer no inhibitor 5 ul 5 ul 5 ul 1X HMT assay buffer 2 20 ul Diluted PRMT1 0 1 0 5 ng ul 20 ul 20 ul 20 ul Total 50 ul 50 ul 50 ul 50 ul 4 Add 5 ul of inhibitor solution of each well designated Test Inhibitor 5 For the Positive Control Substrate Control and Blank add 5 ul of the same solution without
8. t our website at www bpsbioscience com 6044 Cornerstone Court West Ste E San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com RELATED PRODUCTS Product Name Catalog Size PRMT1 expressed in E coli 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 PRMT4 expressed in Sf9 cells 51044 20 ug PRMT5 expressed in HEK293 51045 20 ug PRMT5 expressed in Sf9 cells 51048 20 ug PRMT6 expressed in HEK293 51046 20 ug PRMT8 expressed in Sf9 cells 51052 20 ug PRMT3 Chemiluminescent Assay Kit 52005 96 reactions PRMT4 Chemiluminescent Assay Kit 52041L 96 reactions PRMT5 Chemiluminescent Assay Kit 52002 96 reactions PRMT6 Chemiluminescent Assay Kit 52046 96 reactions Histone H4 R3 Universal Assay Kit 52074 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 140623 140623 6044 Cornerstone Court West Ste E Bioscience TROUBLESHOOTING GUI
9. tion 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 Use different concentrations of linear range of the enzyme PRMT1 BPS Bioscience assay 51040 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
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