Transcription Factor ELISA Kit
Contents
1. Assay Plate a Add 200 uL well of 1X Wash Buffer b Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly c Remove excess wash buffer by inverting the plate and tapping firmly on a clean paper towel d Repeat twice for a total of 3 washes IMPORTANT To avoid cross contamination do not touch pipet tips to the Assay Plate or liquid within the Assay Plate Detecting the To detect the signal Page 12 Signal Step Action 1 Add 100 uL well of Substrate Solution to the Assay Plate 2 Incubate the Assay Plate at room temperature for 5 15 minutes in the dark until there is a medium blue color developed in the wells Do not overdevelop as this will increase the background signal 3 Add 100 uL well of Stop Solution to the Assay Plate The color should change from blue to yellow 4 Head the plate in the spectrophotometer at 450nm within 5 minutes of adding the Stop Solution Transcription Factor ELISA Kit User Manual Troubleshooting Possible Problems and Recommended Solutions Troubleshooting Observation Possible Cause Recommended Action Weak or no signal Reagents not added in correct order Follow instructions in the user manual for performing the assay Presence of assay inhibitors For example sodium azide inhibits HRP reaction We recommend only using the buffers provided in the kit Incorrect spectrophotometer2. NA complexes continued Step Action 3 If a negative control is desired cold probe can be used to confirm competitive binding of the labeled probe Combine the following reagents as such e 10 ul Binding Buffer e 2 5 uL TF Specific Probe e 10 ul TF Specific Cold Probe e 17 5 ul nuclease free water 40 pL Total per well 4 Dispense 40 uL of Binding Buffer Master Mix to each well of the Sample Plate If running a negative control dispense 40 uL from the mixture made in step 3 5 Add 10 uL of the Positive Control provided in the kit to the positive control and negative control wells 6 Prepare samples and add them to the Sample Plate a Dilute nuclear extracts to 0 5 2 0 ug uL using Nuclear Extract Dilution Buffer b Add 10 uL of the diluted extracts to the appropriate wells of the Sample Plate 7 Prepare blank wells by adding 10 pL of Nuclear Extract Dilution Buffer to the appropriate Sample Plate wells 8 Seal the Sample Plate using a Plate Seal and incubate at room temperature for 30 minutes rocking the plate gently at 150 rpm Capturing TF DNA To capture TF DNA complexes Complexes Step Action 1 Wash the Assay Plate a Add 200 uL well of 1X Wash Buffer b Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly c Dry the surface by tapping the inverted plate firmly on a clean paper towel d Repeat twice for a total of 3 washes IMPORTANT To avoid cross con
3. TF ELISA Kit from Panomics to this Manual quantitate the binding activity of a specific TF from nuclear extracts What this Manual This manual provides the following Covers Kit contents and storage conditions e Assay procedures e Troubleshooting Safety Warnings CAUTION All chemicals should be considered potentially hazardous We recommend that and Precautions this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice CAUTION This kit contains small quantities of sodium azide Sodium azide reacts with lead and copper plumbing to form explosive metal azides When disposing flush drains with a large volume of water to prevent azide accumulation Observe all state and local regulations for disposal Note This product is intended for research use only It is not for diagnosis of disease in humans or animals For More For information about the products mentioned in this manual visit our website at Information www panomics com Transcription Factor ELISA Kit User Manual Page 5 About the Transcription Factor TF ELISA Kit About the Transcription Factor TF ELISA Kit Fundamentals of Panomics TF ELISA Kits Assay Overview Page 6 Panomics TF ELISA Kits measure the activity of specific transcription factors in nuclear extracts The assay is highly specific and precise requires a minimum of 5 10 ug of protein well and can be
4. Transcription Factor ELISA Kit User Manual Gh Panomics Panomics Inc Transcription Factor ELISA Kit User Manual Copyright Copyright 2008 Panomics Inc All rights reserved Trademarks Citing in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the TF ELISA target specific Kit For example if a paper cites the TF ELISA NF B p50 Kit product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Disclaimer Panomics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Contents About the User Manual 0 0 0 rava avvik eee ees 5 Who Should Read this Manual llle 5 What this Manual Covers 2 0 0 0 0c n anne 5 Safety Warnings and Precautions 0 000 ee eee 5 For More Information v2 4 d REESE Back a eee a 5 About the Transcription Factor TF ELISA Kit 2 0 0 0 0 0 0 ccc e
5. e eee 6 Fundamentals of Panomics TF ELISA Kits llle 6 ASSay OVGWICW se een Winnie 6 Panomics Transcription Factor ELISA Kit Contents and Storage Conditions 7 Kit Contents and Storage raaav vnr arr ee nenn 7 Required Equipment and Materials Not Provided 22 222 rar eee eee 7 EIE EY See Ra ee Gu Sidhe KANG 7 Materials uer Se oat Soe 8 Guidelines for Assay Design and Analysis llle sn 9 Preparing Samples 4 3 m EE Rr exem oue ee Soe US bed wee 9 General Guidelines llli 9 Analysis OT FeSHISs 5 1 2 dini aske salsa dp rad Det D ert seeks arabe oed 9 ASSaV PIDEBOUELO S oa m anatibo Be aie hod en Veni asa Sod ogee Se e banana ae 9 BEIOrE VS ST 9 Forming TF DNA Complexes aaa 9 Capturing TF DNA Complexes l l 10 Binding Primary and Secondary Antibodies liliis llus 10 Detecting the Signal llle 12 TOUBDIESNOOUNG tes 34 38 28 221 5 00 0125 172 5 1501255 50 at a ees a 13 Possible Problems and Recommended Solutions 005 13 Contacting Pateml6es esasa 5 c Brian 13 TECHNICAL HEP suser dos a caca d dier tad eek ee BEd Die qn itin 13 For Additional Services 0 0 0 ccc eee eee 13 Appendix l Blank Plate Map 15 22 EN oc eee den 14 Transcription Factor ELISA Kit User Manual iii About the User Manual About the User Manual Who Should Read Anyone that has purchased a Transcription Factor
6. ied If stored properly reagents have a shelf life of 6 months IMPORTANT Avoid repeated freeze thaw cycles of nuclear extract TF ELISA Kit components Component Description Storage Location Positive Control Nuclear extract or recombinant protein for positive assay control 80 C Box 1 of specific target TF Specific Probe Biotin labeled double stranded oligonucleotide with consensus 20 C Box 1 sequences for specific TF TF Specific Cold Non labeled double stranded oligonucleotide corresponding 20 C Box 1 Probe with probe Cold probe is optional as a negative control to measure competition of the labeled probe Binding Buffer Aqueous buffered solution for TF binding 20 C Box 1 Antibody Dilution Aqueous buffered solution for diluting antibody 20 C Box 1 Buffer Nuclear Extract Aqueous buffered solution for diluting nuclear extract 20 C Box 1 Dilution Buffer Primary Antibody 200x antibody recognizing the specific TF protein 4 C Box 2 Secondary Antibody 200x IgG HRP conjugated antibody specific to the IgG of the 4 C Box 2 primary antibody 10x Wash Buffer Aqueous buffered solution for washing off non specific binding 4 C Box 2 Substrate Solution Chromogenic reagents for HRP 4 C Box 2 Stop Solution Aqueous solution for stopping the chromogenic reaction 4 C Box 2 Plate Seal Clear plastic seals for sealing plates during the assay 15 30 C Box 2 Assay Plate 96 well clear s
7. performed in 4 hours For a complete list of available ELISA Kits visit www panomics com All assays are performed on the 96 well plate The illustration uses NF B p50 as the transcription factor example TMB HRP 2nd H 1st Ab hr NFkBp50 We Biotin Streptavidin Coated Plate Activated NF B p50 molecules from nuclear extracts bind to an NF B p50 consensus binding site NF B p50 Probe on a biotinylated oligonucleotide These oligonucleotides are then immobilized on a streptavidin coated 96 well plate The NF B p50 bound to the oligonucleotide is detected by an antibody directed against NF B p50 An additional horseradish peroxidase HRP conjugated secondary antibody reacts with the TMB substrate to provide a sensitive colorimetric readout which is quantified by spectrophotometry TMB or tetramethylbenzidine is a chromogen that yields a blue color when oxidized with hydrogen peroxide catalyzed by HRP with major absorbances at 370 nm and 652 nm The color then changes to yellow with the addition of a stop solution phosphoric acid with maximum absorbance at 450 nm Transcription Factor ELISA Kit User Manual Panomics Transcription Factor ELISA Kit Contents and Storage Conditions Panomics Transcription Factor ELISA Kit Contents and Storage Conditions Kit Contents and Storage The TF ELISA Kit contains the following components Refer to the product insert for quantities and details of components suppl
8. settings Insufficient protein Check to make sure the wavelength is 450 nm Increase the amount of protein in the nuclear extract We recommend preparing nuclear extracts with Panomics Nuclear Extraction Kit P N AY2002 Target protein not activated induced Heview induction procedures You may need to change cell lines inducer or induction conditions High background Samples overdeveloped Incorrect quantities of antibody or wash buffer was used Shorten the development time Check to make sure dilutions were performed correctly all wells are filled with wash buffer during wash steps and residual wash buffer removed by inverting plate on a paper towel Contacting Panomics Technical Help For Additional Services Transcription Factor ELISA Kit User Manual For technical questions contact our technical support group by telephone at 1 877 726 6642 option 3 or by email at techsupport panomics com US and Canada For technical support in Europe email techsupport_europe panomics com You can also visit our website www panomics com for an updated list of FAQs and product support literature For information about Panomics products or for ordering information contact your Regional Sales Manager or visit our website at www panomics com Page 13 Appendix I Blank Plate Map Appendix I Blank Plate Map Page 14 Transcription Factor ELISA Kit User Manual
9. ssay Plate Seal the Assay Plate with a Plate Seal and incubate at room temperature for 1 hour rocking the plate gently at 150 rpm Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly Wash the Assay Plate a Add 200 uL well of 1X Wash Buffer b Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly c Remove excess wash buffer by inverting the plate and tapping firmly on a clean paper towel d Repeat twice for a total of 3 washes IMPORTANT To avoid cross contamination do not touch pipet tips to the Assay Plate or liquid within the Assay Plate Prepare fresh Secondary Antibody by diluting it 1 200 using Antibody Dilution Buffer then invert to mix For example for one 96 well plate mix 50 uL of Secondary Antibody with 9950 uL of Antibody Dilution Buffer Add 100 uL well of the diluted Secondary Antibody to the Assay Plate 10 11 Seal the Assay Plate with a Plate Seal and incubate at room temperature for 1 hour rocking the plate gently at 150 rpm Note At this point remove the Substrate Solution and Stop Solution from 4 C and equilibrate to room temperature Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly Transcription Factor ELISA Kit User Manual Page 11 Assay Procedure To bind primary and secondary antibodies continued Step Action 12 Wash the
10. tained using a TECAN Phenix GENios at 450 nm other instruments may give different results The ratio of the provided positive control to the blank well should be 2 5 or higher The ratio of positive control to the competitive control cold probe should be 2 0 or higher For best results follow the instructions that accompany the spectrophotometer Assays CVs CV std dev mean x 100 are typically less than 15 for technical replicates Assay Procedure Before You Start Forming TF DNA Complexes e Thaw Positive Control and sample extracts on ice e Thaw TF Specific Probe and TF Specific Cold Probe on ice e Prepare 1X Wash Buffer by diluting 60 mL of 10X Wash Buffer into 540 mL of nuclease free sterile water Note 1X Wash Buffer is good for 6 months at 4 C To form TF DNA complexes Step Action 1 Using the blank plate map in Appendix I prepare an experimental plate map for the Sample Plate designating which wells are samples positive controls blank wells and competition controls 2 Prepare a Binding Buffer Master Mix Each well of the Sample Plate requires 40 uL of Binding Buffer Master Mix Combine the following reagents multiply the volumes by the number of wells you are running 10 ul Binding Buffer e 2 5 uL TF Specific Probe e 27 5 ul nuclease free water 40 uL Total per well x Number of wells Transcription Factor ELISA Kit User Manual Page 9 Assay Procedure To form TF D
11. tamination do not touch pipet tips to the Assay Plate or liquid within the Assay Plate Note This wash step and subsequent wash steps can be performed using a plate washer if desired 2 Transfer 45 uL from each well of the Sample Plate to each well of the Assay Plate 3 Seal the Assay Plate with a Plate Seal and incubate at room temperature for 1 hour rocking the plate gently at 150 rpm Binding Primary To bind primary and secondary antibodies and Secondary Page 10 Antibodies Step Action 1 Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly Transcription Factor ELISA Kit User Manual Assay Procedure To bind primary and secondary antibodies continued Step Action 2 Wash the Assay Plate a Add 200 uL well of 1X Wash Buffer b Invert the Assay Plate over an appropriate receptacle and expel the contents forcibly c Remove excess wash buffer by inverting the plate and tapping firmly on a clean paper towel d Repeat twice for a total of 3 washes IMPORTANT To avoid cross contamination do not touch pipet tips to the Assay Plate or liquid within the Assay Plate Prepare fresh Primary Antibody by diluting it 1 200 using Antibody Dilution Buffer then invert to mix For example for one 96 well plate mix 50 uL of Primary Antibody with 9950 uL of Antibody Dilution Buffer Add 100 uL well of the diluted Primary Antibody to the A
12. treptavidin coated plate with 12 strips and white 15 30 C Box 2 12 strips plastic holder Sample Plate 96 well v bottom plate 15 30 C Box 2 Required Equipment and Materials Not Provided Equipment Item Source TECAN Phenix GENios model F129015 or equivalent Microplate Spectrophotometer with 450 nm filter Rocking platform VWR P N 40000 300 Bio Rad ImmunoWash model 1575 or equivalent Plate washer optional Transcription Factor ELISA Kit User Manual Page 7 Required Equipment and Materials Not Provided Materials Item Source Reagent Reservoirs 25 mL and 100 mL capacities Diversified Biotech P N RESE 3000 RESE 1000 Nuclear Extraction Kit Panomics P N AY2002 Nuclease free sterile water Major laboratory supplier Page 8 Transcription Factor ELISA Kit User Manual Guidelines for Assay Design and Analysis Guidelines for Assay Design and Analysis Preparing Samples General Guidelines Analysis of Results Protein concentration of sample inputs should be at least 0 5 2 ug uL e Read this user manual and all product inserts before performing the assay e Store all reagents at the recommended temperatures e Use Panomics Nuclear Extraction Kit for best results Note The Assay Plate contains 12 strips which you can use separately The positive control should generate an absorbance above 0 5 The blank wells should generate an absorbance below 0 2 These values were ob
Download Pdf Manuals
Related Search