Data Sheet - BPSBioscience.com
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1. 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 wells Background signal to noise ratio is high Insufficient washes Increase number of washes Increase wash volume Increase Tween 20 concentration to 0 1 in PBST 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 TNKS1 BPS Bioscience 80504 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 120411 Final2. 6044 Cornerstone Court W Ste E San Diego CA 92121 M J Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet TNKS1 Histone Ribosylation Assay Kit Antibody Detection Catalog 80575 Size 384 reactions DESCRIPTION The TNKS1 Histone Ribosylation Assay Kit Antibody Detection is designed to measure Tankyrase 1 activity using purified Tankyrase 1 TNKS1 or cell extracts containing TNKS1 The TNKS1 Histone Ribosylation Assay Kit Antibody Detection comes in a convenient format with a 384 well plate precoated with histone mixture antibody against poly ADP ribose modified histone the secondary HRP labeled antibody NAD PARP assay buffer and purified TNKS1 enzyme for 384 enzyme reactions The key to the TNKS1 Histone Ribosylation Assay Kit Antibody Detection is a highly specific antibody that recognizes poly ADP ribose modified histone With this kit only three simple steps on a microtiter plate are required for Tankyrase 1 reaction First NAD is incubated with a sample containing assay buffer and enzyme for one hour 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 be measured using a chemiluminescence reader Note This kit is also suitable for use with cell extracts but non specific ribosylation activity may be detected CO
3. MPONENTS Catalog Reagent Amount Storage 80504 TNKS1 4 ug 80 C NAD 500 ul 80 C 10x PARP Assay Buffer 2ml 20 C 52140K Primary antibody 11 26 ul 80 C 52130H Secondary HRP labeled antibody 1 20 ul 80 C Avoid Blocking Buffer 160 ml 4 C freeze thaw HRP chemiluminescent substrate 2 A cycles components 12 mleach 4 C 384 well plate pre coated with o aaen pits we MATERIALS REQUIRED BUT NOT SUPPLIED PBST buffer 1x PBS containing 0 05 Tween20 Luminometer or fluorescent microplate reader capable of reading chemiluminescence Adjustable micropipettor and sterile tips Rotating or rocker platform Paper towels 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 120411 Final 6044 Cornerstone Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com 2 Bioscience APPLICATIONS Great for studying enzyme kinetics screening small molecular inhibitors for drug discovery and HTS applications CONTRAINDICATIONS DMSO gt 1 strong acids or bases ionic detergents high salt STABILITY 6 months 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 samp
4. PS Bioscience 80574 80575 Luminescence was measured using a Bio Tek fluorescent microplate reader Data shown is lot specific For lot specitic 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 120411 Final 6044 Cornerstone Court W Ste E a n 7 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 TNKS2 Histone Ribosylation Assay Kit 80577 384 rxns TNKS1 Histone Ribosylation Assay Kit Biotin labeled NAD 80573 96 rxns TNKS1 Histone Ribosylation Assay Kit Antibody Detection 80574 96 rxns TNKS2 Histone Ribosylation Assay Kit 80576 96 rxns PARP1 Chemiluminescent Assay Kit 80551 96 rxns PARP2 Chemiluminescent Assay Kit 80552 96 rxns PARP3 Chemiluminescent Assay Kit 80553 96 rxns PARP6 Chemiluminescent Assay Kit 80556 32 rxns PARP7 Chemiluminescent Assay Kit 80557 96 rxns PARP11 Chemiluminescent Assay Kit 80561 96 rxns PARP1 Enzyme 80501 10 ug PARP2 Enzyme 80502 10 ug PARP3 Enzyme 80503 10 ug PARP6 Enzyme 80506 10 ug TNKS1 PARP5A Enzyme 80504 10 ug TNKS2 PARP5B 667 end Enzyme 80505 10 ug TNKS2 PARP5B 849 end Enzyme 80515 10 ug PARP7 Enzym
5. 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 12041 1Final 6044 Cornerstone Court W Ste E San Diego CA 92121 M J Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com 7 Add 50 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 11 800 fold with Blocking buffer 2 Add 50 ul per well Incubate 1 hour at room temperature with slow shaking 3 Wash plate with PBST buffer and Blocking buffer as in steps 1 6 and 1 7 Dilute Secondary HRP labeled antibody 1 1 000 fold with Blocking buffer Add 50 ul per well Incubate for 30 min at room temperature with slow shaking Wash plate with PBST buffer and Blocking buffer as in steps 1 6 and 1 7 Just before use mix on ice 25 ul HRP chemiluminescent substrate A and 25 ul HRP chemiluminescent substrate B and add 50 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 TNKS1 XAV939 120 100 IC50 6 5NnM Activity oa o 1 0 0 5 0 0 0 5 1 0 1 5 2 0 XAV939 Log nM Inhibition of TNKS1 by XAV939 measured using the TNKS1 Histone Ribosylation Assay Kit Antibody Detection B
6. e 80507 10 ug PARP9 Enzyme 80509 10 ug PARP11 Enzyme 80511 10 ug PARP12 Enzyme 80512 10 ug 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 120411Final Bioscience TROUBLESHOOTING GUIDE Problem Possible Cause 6044 Cornerstone Court W Ste E San Diego CA 92121 Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com Solution Luminescence signal of positive control reaction is weak TNKS1 enzyme has lost activity Enzyme loses activity upon repeated freeze thaw cycles Use fresh enzyme TNKS1 BPS Bioscience 80504 Store enzyme in single use aliquots Increase time of enzyme incubation Increase enzyme concentration Antibody reaction is insufficient Increase time for primary antibody incubation Avoid freeze thaw cycles of antibodies Incorrect settings on instruments Record light signals at 5 second intervals Refer to instrument instructions for settings to increase sensitivity of light detection Chemiluminescent reagents mixed soon too Chemiluminescent solution should be used within 15 minutes of mixing Ensure both reagents are properly mixed Luminescent erratic or among wells signal is varies widely Inaccurate pipetting technique
7. les and controls should be tested in duplicate Step 1 1 Rehydrate the microwells by adding 50 ul of PBST buffer 1 x PBS 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 TNKS1 enzyme on ice Upon first thaw briefly spin tube containing enzyme to recover full content of the tube Aliquot TNKS1 enzyme into single use aliquots Store remaining undiluted enzyme in aliquots at 80 C Note TNKS1 enzyme is very sensitive to freeze thaw cycles Do not re use thawed aliquots or diluted enzyme 3 Dilute TNKS1 enzyme in 1X PARP assay buffer at 1 ng ul 10 ng 10 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 Substrate Blank Control Sample Control TNKS1 1 ng ul 10 ul 10 ul 10 ul 10X PARP Assay Buffer 2 5 ul 2 5 ul 2 5 ul 2 5 ul NAD 1 25 ul 1 25 ul 1 25 ul Test Inhibitor Activator X ul H2O 11 25 ul 11 25 X 12 5 ul 21 25 ul ul Total 25 ul 25 ul 25 ul 25 ul 5 Add the entire reaction mixture 25 ul to the substrate coated plate Incubate at room temperature for 1 hour 6 Wash the plate three times with PBST buffer Blot dry onto clean paper towels OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE
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