Data Sheet - BPSBioscience.com
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1. Sample Incubate at room temperature for 1 hour 11 Remove the supernatant from the wells and wash the strip three times with 200 ul TBST buffer Blot dry onto clean paper towels 12 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 1 100 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 11 and 1 12 Step 3 1 Dilute Secondary HRP labeled antibody 1 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 11 and 1 12 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 140411 140411 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 4 Just before use mix on ice 50 ul HRP chemiluminescent substrate A2. 0 ul 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 140411 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 Add 5 ul of inhibitor solution of each well designated Test Inhibitor 6 For the Positive Control Substrate Control and Blank add 5 ul of the same solution without inhibitor inhibitor buffer 7 Thaw SUV39H1 enzyme on ice Upon first thaw briefly spin tube containing enzyme to recover full contents of the tube Aliquot SUV39H1 enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C immediately Note SUV39H1 enzyme is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 8 Dilute SUV39H1 enzyme in 1x HMT assay buffer 1 to 5 ng ul 100 ng 20 ul Keep diluted enzyme on ice until use Discard any unused diluted enzyme after use Note Diluted enzyme may not be stable Dilute enzyme immediately before use 9 Add 20 ul of 1x HMT buffer 1 to the well designated Blank 10 Initiate reaction by adding 20 ul of diluted SUV39H1 enzyme to the wells designated Positive Control Substrate Control and Test
3. 1 100 ul 80 C 52130H Secondary HRP labeled antibody 1 10 ul 80 C 52160 4x HMT assay buffer 1 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 Tween 20 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 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 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 S C et al Genome Biology 2005 6 227 ASSAY PROTOCOL All samples and controls should be tested in duplicate Step 1 1 Rehydrate
4. 140411 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 SUV39H1 Chemiluminescent Assay Kit Catalog 52006L Size 96 reactions DESCRIPTION The SUV39H1 Chemiluminescent Assay Kit is designed to measure SUV39H1 activity for screening and profiling applications The SUV39H1 Chemiluminescent Assay Kit comes in a convenient format with 8 well strips precoated with histone H3 peptide substrate primary antibody against methylated lysine residue of Histone H3 the secondary HRP labeled antibody S adenosylmethionine methyltransferase assay buffer and purified SUV39H1 enzyme for 96 enzyme reactions The key to the SUV39H1 Direct Activity Assay Kit is a highly specific antibody that recognizes methylated K9 residue of Histone H3 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 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 51070 SUV39H1 human enzyme 10 ug 80 C 52120 100 uM S adenosylmethionine 250 ul 80 C 52140A Primary antibody
5. 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 reader 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 wavelength 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 Do not use a filter when measuring light emission Optimal settings will vary depending on the particular plate reader Typical integration time is 1 second delay after plate movement is 100 msec Typical settings for the Synergy 2 Bio Tek plate reader are use the hole position on the filter wheel Optics position Top Read type endpoint Sensitivity may be adjusted based on luminescence of a control assay without enzyme typically we set this value as 100 Example of Assay Results 10000 8000 6000 4000 Luminescence 2000 0 25 50 75 100 125 SUV39H1 ng SUV39H1 enzyme activity measured using the SUV39H1 Chemiluminescent Assay Kit BPS Bioscience Catalog 52006L Luminescence was measured using a Bio Tek fluorescent microplate reader Data shown is lot specific For lot
6. ioscience 51070 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
7. me loses activity upon repeated freeze thaw cycles Use fresh enzyme SUV39H1 BPS Bioscience 51070 Store enzyme in single use aliquots Increase time of enzyme incubation Increase enzyme concentration Antibody reaction is Increase time for antibody incubation insufficient Avoid freeze thaw cycles of antibody Incorrect settings on Refer to instrument instructions for instruments settings to increase sensitivity of light detection Refer to the section Reading Chemiluminescence 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 the 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 range of the assay Use different concentrations of enzyme SUV39H1 BPS B
8. 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 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 SUV39H1 82 end enzyme 51070 50 ug SUV39H1 full length enzyme 51071 5 ug SUV39H2 enzyme 51080 50 ug SUV39H2 Chemiluminescent Assay Kit 52008 96 reactions H3 K9 Universal Methyltransferase Assay Kit 52072 96 reactions G9a enzyme E coll 51000 50 ug G9a enzyme Sf9 cells 51001 20 ug G9a Homogeneous Assay Kit 52051 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 140411 140411 6044 Cornerstone Court West Ste E Bioscience TROUBLESHOOTING GUIDE Problem Luminescence signal of positive control reaction is weak Possible Cause SUV39H1 enzyme has lost activity San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Enzy
9. 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 strip plate onto clean paper towels to remove liquid 2 Thaw S adenosylmethionine on ice Upon first thaw briefly spin the 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 Dilute 100 uM S adenosylmethionine 4 fold with water to make a 25 uM solution Dilute only the amount of S adenosylmethionine required for the assay Discard any unused diluted S adenosylmethionine after use 4 Prepare the master mixture N wells x 7 5 ul 4x HMT assay buffer 1 5 ul diluted 25 uM S adenosylmethionine 12 5 ul H20 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 1 17 5 ul water Substrate Positive Test Blank Control Control Inhibitor 4x HMT assay buffer 1 7 5 ul 7 5 ul 7 5 ul 7 5 ul 25 uM S adenosylmethionine 5 ul 5 ul 5 ul H2O 12 5 ul 17 5 ul 12 5 ul 12 5 ul Test Inhibitor z 5 ul Inhibitor buffer no inhibitor 5 ul 5 ul 5 ul 1x HMT assay buffer 1 20 ul SUV39H1 5 ng ul 20 ul 20 ul 20 ul Total 50 ul 50 ul 50 ul 5
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