Procarta™ Transcription Factor Assay Kit
Contents
1. C Protease Inhibitor Aqueous solution for inhibiting protease activity 20 C Cocktail Binding Buffer Aqueous buffered solution for protein DNA 2 8 C binding Procarta Transcription Factor Assay Kit User Manual Page 7 Required Materials and Equipment Not Provided Procarta Transcription Factor Assay Kit components continued Component Description Storage Separation Buffer Aqueous buffered solution for separation of 2 8 C protein DNA complex from free probes Assay Buffer Aqueous buffered solution for hybridization 2 8 C Wash Buffer Aqueous buffered solution for Capture Bead 2 8 C washing Reading Buffer Aqueous buffered solution for detection of 2 8 C Capture Beads Capture Beads premixed Pre mixed beads conjugated with oligo 2 8 C Dark corresponding to the Detection Probes Streptavidin PE Streptavidin conjugated R Phycoerythrin for 2 8 C Dark detection of bound biotin probes Elution Buffer Aqueous buffered solution for eluting the 15 30 C bound TF probes from protein PCR Plate 96 well clear PCR plate for Protein DNA 15 30 C Complex formation Sample Collection Plate 96 well clear skirted PCR plate 15 30 C Utility Plate 96 well clear polystyrene plate for collection of 15 30 C waste during the separation of TF Bound Detection Probes Also used as a holder for the Filter Plate during the detection of eluted Detection Probes Separation Plate 96 wel2. a whole plate trim a PCR Plate Seal to the appropriate size and seal any unused wells of the PCR Plate 3 Prepare the PCR Plate a Add 10 uL of Detection Probe to all experimental wells of the PCR Plate b Add 10 pL of Nuclear Extract Controls and or Whole Cell Lysate Controls to the appropriate assay wells c Add 10 uL of Working Sample Dilution Buffer to assay background control wells d Add 10 uL of prepared sample to each designated well Note As a starting point we recommend that each 10 uL sample contain a total of 2 ug of protein Dilute samples with Working Sample Dilution Buffer Procarta Transcription Factor Assay Kit User Manual Page 13 Assay Procedure To form Protein DNA complexes continued Step Action 4 Mix samples by gently tapping the bottom of the PCR Plate IMPORTANT Make sure all bubbles are removed from the wells Tapping the plate should accomplish this 5 Incubate the plate at 15 C for 30 minutes IMPORTANT Make sure the temperature is maintained at 15 C We recommend using a PCR instrument Samples can be kept at 4 C for up to 1 hour before proceeding to the next step Eluting TF Bound To elute TF bound detection probes Detection Probes Page 14 Step Action 1 Prepare Separation Plate a Trim a foil Plate Seal to the appropriate size and seal any unused wells of the Separation Plate b Place the Utility Plate on the bottom of the Separ
3. cee LL LL La 13 Eluting TF Bound Detection Probes Qu La LL LL 14 Denaturing the Eluted Detection Probes 15 Hybridizing the Denatured Detection Probes 16 Binding the SAPE ui a RUD ee ae Era ete Be ee alae ED ER dom RE 17 Detecting ME signal seria ito e ec e a ari tant PE E 17 Troubleshooting is 2433 picto bog buc Sige es EN keh eas Pup ea S do ied 19 Possible Problems and Recommended Solutions Ls 19 Contacting Panoml6Ss ss RS E ot atio bee tek e ees Co tob tad ty Bate iN a 20 TECNICA AGH seis Beb s a is Ree cete EUR aa ek ded io oben n owed 20 For Additional oerVIGeS aan ut Sa hieu o sac weed Dat berg 20 Procarta Transcription Factor Assay Kit User Manual lii Table of Contents Appendix Bead Analyte Associations Appendix Sample and Blank Plate Layouts Procarta Transcription Factor Assay Kit User Manual About the User Manual About the User Manual Who Should Read Anyone that has purchased a Procarta Transcription Factor TF Assay Kit from this Manual Panomics to perform profiling of up to 40 different transcription factors per reaction in the following sample types Nuclear extracts from cultured cells Cell lysates from cultured cells What this Manual This manual provides recommendations and step by step procedures for the Covers following Guidelines for as
4. foil Shake the assembly on a plate shaker at room temperature for 30 minutes at 300 500 rpm o 29 Tp Detecting the To detect the signal Signal 9 Step Action 1 Prepare to wash the Capture Beads a Remove the Plate Seal carefully to avoid splashing of samples b Remove the buffer using the vacuum manifold c Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Wash the Capture Beads a Dispense 150 uL of Wash Buffer to each well of the Filter Plate b Remove the Wash Buffer using the vacuum manifold c Repeat step 2a 2b once more d Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Resuspend Capture Beads a Dispense 120 uL of Reading Buffer to each well of the Filter Plate b Seal the wells with a Plate Seal c Wrap the Filter Plate Utility Plate assembly with aluminum foil d Shake the Filter Plate at 300 500 rpm and room temperature for 5 minutes or until ready to read on the Luminex instrument Note The Filter Plate Utility Plate assembly can be wrapped with aluminum foil and stored flat in the dark at 4 C for up to 48 hours before proceeding However delay in reading the plate may result in decreased sensitivity for some analytes Shake the plate at 300 500 rpm for 5 minutes before reading Procarta Transcription Factor Assay Kit User Manual Page 17 Ass
5. x 100 Assay CVs are typical less than 15 for technical replicates Fold Change Subtract average MFI background from all samples Calculate fold change as the treated sample value untreated sample value Assay Controls For the positive control calculate the ratio of the NF B signal MFI divided by the assay background For NF B this value should be gt 20 For each set of positive and negative controls subtract the NF B assay background from the NF B signals in the controls Then divide the background corrected NF B signal from the positive control by the background corrected NF B signal from the negative control This ratio or fold change calculation should be gt 5 Procarta Transcription Factor Assay Kit User Manual Page 11 Set Up and Operation of the Vacuum Manifold System Set Up and Operation of the Vacuum Manifold System About Using the Vacuum Manifold Sealing Filter Plates Setting Up and Calibrating the Manifold Page 12 This topic describes how to set up and use the Millipore vacuum manifold This includes how to calibrate the pressure and important guidelines that will help to ensure good assay reproducibility We recommend that you set up and calibrate the manifold before you start the assay to ensure the assay is performed without interruption Lay a Plate Seal over the Filter Plate and roll a 5 mL serological pipet or equivalent over the Plate Seal to seal the Filter Plate This ensu
6. ANT Do not seal too tight as this may cause leaking c Place the Filter Plate on top of the Utility Plate Incubate the Filter Plate Utility Plate assembly in a Vortemp shaking incubator set at 50 C and 300 500 rpm for 30 minutes Note Ifa shaking incubator is not available wrap the Filter Plate Utility Plate assembly with aluminum foil shake the assembly at room temperature for 10 minutes then transfer to a 50 C incubator for 30 minutes without shaking Proceed to the next step Binding the SAPE on page 17 Page 16 Procarta Iranscription Factor Assay Kit User Manual Assay Procedure Binding the SAPE To bind the SAPE Step Action 1 Prepare to wash the Capture Beads a Remove the Plate Seal carefully to avoid splashing of samples b Remove the buffer using the vacuum manifold c Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Wash the Capture Beads a Dispense 150 uL of Wash Buffer to each well of the Filter Plate b Remove the Wash Buffer using the vacuum manifold c Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Add the Streptavidin PE Invert the Streptavidin PE tube to mix Dispense 100 uL of Streptavidin PE to each well of the Filter Plate Seal the Filter Plate with a foil Plate Seal Wrap the Filter Plate Utility Plate assembly with aluminum
7. Procarta Transcription Factor Assay Kit User Manual Gh Panomics Panomics Inc Procarta Transcription Factor Assay Kit User Manual Copyright Copyright 2006 Panomics Inc All rights reserved Trademarks Procarta is a trademark of Panomics Inc Citing Procarta in Publications When describing a procedure for publication using this product we would appreciate it if you would refer to it as the Procarta Transcription Factor Assay Kit If a paper cites a Procarta 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 41 220824 2 a ad aa Osea KS eae A weeks 5 Who Should Read this Manual LL LL ee 5 What this Manual COVES 35 5 a ine ea 5 Safety Warnings and Precautions Lu LL LL LL LL Le 5 For More Information LL LL LL LL 5 About t
8. and then filter the solution Avoid splashing and cross contamination of wells during all wash steps IMPORTANT During filtration maintain the vacuum between 2 3 mm of Hg Higher vacuum settings may result in loss of Capture Beads IMPORTANT Do not allow the Filter Plates to air dry following washes Immediately add the next component following each filtration step 3 Break the vacuum immediately after each solution has been completely filtered from all wells approximately 2 5 seconds by first turning off the vacuum then removing the plate from the manifold Note Wells typically filter at different rates 4 Place the Filter Plate back on the Utility Plate 5 Following the last wash in each series blot the bottom of the Filter Plate thoroughly with a paper towel to remove traces of Wash Buffer Avoid touching the bottom of the Filter Plate with your fingers or to the bench during manipulations Assay Procedure Before You Start 4 Prepare Working Sample Dilution Buffer Based on your dilution calculations prepare a Working Sample Dilution Buffer by combining 100 parts of Sample Dilution Buffer 1 part DTT and 1 part Protease Inhibitor Cocktail Store on ice Place Binding Buffer on ice 4 If Elution Buffer contains precipitates warm to 37 C with gently swirling Forming To form Protein DNA complexes Protein DNA E XS Complexes ep Action 1 Thaw Detection Probes on ice 2 If you are not using
9. anual Page 15 Assay Procedure To denature the eluted Detection Probes continued Step Action 6 Proceed to the next step Hybridizing the Denatured Detection Probes on page 16 Hybridizing the To hybridize the denatured Detection Probes Denatured Detection Probes SteP Action 1 Add premixed Capture Beads to the Filter Plate a Vortex the premixed Capture Bead solution at the highest setting for 30 seconds b Dispense 50 uL of premixed Capture Beads to each well of the Fliter Plate Remove the Capture Bead buffer using the vacuum manifold d Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate IMPORTANT Do not invert or tap the Filter Plate Wash the Capture Beads a Dispense 150 uL of Wash Buffer to each well of the Filter Plate b Remove the Wash Buffer using the vacuum manifold c Blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Dispense 40 uL of Assay Buffer to each well of the Filter Plate Add denatured Detection Probes from Eluting TF Bound Detection Probes on page 14 a Transfer 20 uL of each eluted sample to each well of the Filter Plate according to your assay plate map IMPORTANT Change the pipet tip after every transfer and avoid creating bubbles b Seal the wells of the Filter Plate with a foil Plate Seal IMPORT
10. aper towel to avoid contaminating the bottom of the Separation Plate e Place the Separation Plate back on top of the Utility Plate Procarta Iranscription Factor Assay Kit User Manual Assay Procedure To elute TF bound detection probes continued Step Action 6 Repeat step 5 omitting step 5b four more times for a total of 5 washes IMPORTANT Do not reduce the number of washes as this will result in high background Centrifuge the Separation Plate Utility Plate assembly at 563 x g for 3 minutes at 4 C Elute TF bound Detection Probes a Add 60 uL of Elution Buffer to the center of each experimental well in the Separation Plate b Seal unused wells of the Sample Collection Plate with a foil Plate Seal c Place the Separation Plate on top of the Sample Collection Plate and incubate at room temperature for 5 minutes d Centrifuge the Sample Collection Separation Plate assembly at 563 x g for 3 minutes You should have 60 uL well in the Collection Plate IMPORTANT Use a scale to balance the Sample Collection Separation Plate assembly Place the samples on ice and continue to the next step Denaturing the Eluted Detection Probes on page 15 or cover with a foil Plate Seal and store at 20 C until you are ready to use Thaw on ice before use Denaturing the IMPORTANT For 1 plate kits there is sufficient reagents to process 1 entire plate using Eluted Detection reagent
11. ation Plate 2 Pre wet the Separation Plate a Add 180 uL well ice cold Binding Buffer to each well of the Separation Plate b Centrifuge the Separation Plate Utility Plate assembly at 563 x g for 2 minutes at 4 C c Discard the flow through and dry the top surface of the Utility Plate with a clean paper towel to avoid contaminating the bottom of the Separation Plate IMPORTANT Do not place the Separation Plate directly on any surface other than the specified plates as this might result in cross contamination of wells Place the Utility Plate on the bottom of the Separation Plate 3 Transfer the samples to the Separation Plate a Add 20 uL ice cold Binding Buffer to each well of the protein DNA complexes in the PCR Plate Avoid pipetting up and down b Transfer the entire 40 uL sample to the corresponding well of the Separation Plate Make sure you apply the sample to the center of the filter 4 With the Utility Plate on the bottom of the Separation Plate incubate on ice for 30 minutes IMPORTANT Do not exceed the incubation time as this will result in high background 5 Wash the Separation Plate a Add 180 uL well ice cold Separation Buffer to each well of the Separation Plate b Incubate Separation Plate Utility Plate assembly on ice for 5 minutes Centrifuge the Separation Plate Utility Plate assembly at 563 x g for 2 minutes at 4 C d Discard the flow through and dry the top surface of the Utility Plate with a clean p
12. ay Procedure To detect the signal continued Step Action 4 Read the plate a Remove the Plate Seal carefully to avoid splashing of samples b Read the plate using the following settings Bead Events Bead Sample Size DD Gate Timeout Region Statistic 50 uL 8 000 15 000 25 seconds 50 Median These settings are for the Luminex 100v 1 7 or the Luminex 100IS v2 1 2 2 Luminex or Bio Plex instruments If using other instruments follow the manufacturer s instructions IMPORTANT Check to ensure that the Luminex instrument probe height needle is adjusted appropriately for the Filter Plate IMPORTANT We recommend you calibrate the Luminex instrument each day you run the assay Note The Bio Plex Suspension Array System allows calibration using Low or High sensitivity settings Select the sensitivity during calibration using pre determined values of CAL2 RP1 target as provided by Bio Rad Laboratories Using RP1 Low target value will provide results comparable to those obtained from the Luminex 100 Using the RP1 High target may increase detection sensitivity for low TF protein concentrations but sacrifice part of the linear dynamic range for high concentrations We recommend RP1 High target value for Bio Plex Page 18 Procarta Transcription Factor Assay Kit User Manual Troubleshooting Troubleshooting Possible Problems sion Possible Cause Recommended Action and Reco
13. ed Sample B 4 ug from WCL treated B NCc NE Sample A 1 ug from NE treated Sample B 2 ug from WCL treated C PC WCL Sample A 0 5 ug from NE treated Sample B 1 ug from WCL treated D NC WCL Sample A 2 ug from NE untreated Sample B 4 ug from WCL untreated E Assay Back Sample A 1 ug from NE untreated Sample B 2 ug from WCL untreated ground F Sample A 0 5 ug from NE untreated Sample B 1 ug from WCL untreated G H a PC Positive Control b NE Nuclear Extract c NC Negative Control d WCL Whole Cell Lysate Page 22 Procarta Transcription Factor Assay Kit User Manual Appendix Il 1 2 3 4 5 6 8 9 10 11 12 Procarta Transcription Factor Assay Kit User Manual Page 23
14. he Procarta Transcription Factor Assay Kit 6 Fundamentals of Procarta Transcription Factor Assay 6 ASSAY OVEIVIEW sis add ad ai EA 6 Available Kit Formats s e AERE EA ee E NE a e a are T Procarta Transcription Factor Assay Kit Contents and Handling Conditions T Kit Contents and Storage eee 7 o AS O TEILT 8 Required Materials and Equipment Not Provided 8 A cada Da cnn ce Pana Ge abn st ter eit ae dl 8 Maternal ar A iS p M UE prr idt El 9 Guidelines for Assay Design and Data Analysis oooooooooooo 10 OVEMMICW 4 oid eg dre a Eu a a deae X e daw Ore BRE Re ea 10 Preparing Samples assez mas ea rey Eun Ss eho ee A BUR UH IS 10 Optimizing Sample INPUT i345 v woe Eis rue ana Et 10 Running Assay Controls 2 2222 aa aaa eee eee 10 Running Replicates un a ie a A ad 10 Analyzno Data eu rs EE ee i ei 11 Set Up and Operation of the Vacuum Manifold System 12 About Using the Vacuum Mani old LL LL LL LL LL oo 12 Sealing Filter Plates sus dee ed tmr eb RE Y Se Oe aw SEE Eu a ea 12 Setting Up and Calibrating the Manifold o o o oooooooooo 12 Operating the Manifold LL LL LL LL LL 13 RSS AV PIOGEQUFE a cargo al a cec esc ola ey ee nn She Dich lees sc eas 13 Before YOU SAT at tdi iii 13 Forming Protein DNA Complexes 00 00
15. l white plate for the separation of 15 30 C Protein DNA complexes from free probes PCR Plate Seal Plastic sealer for sample collection plate used 15 30 C to denature samples at 95 C and other PCR plates Plate Seal Adhesive backed foil for sealing filter plates 15 30 C during bead assay Filter Plate 96 well sterile filter plate 15 30 C Kit Handling 4 Store all controls at 80 C avoid multiple freeze thaws If precipitates occur in the Elution Buffer warm to 37 C with gentle swirling Required Materials and Equipment Not Provided Equipment Page 8 Item Vacuum filtration system Source Millipore P N MAVMO960R and WP6111560 PCR Instrument for controlled incubation of samples in PCR Plates MJ Research Model PCT 200 or equivalent Microplate centrifuge capable of working at 4 C equivalent Eppendorf 5415D or Procarta Transcription Factor Assay Kit User Manual Required Materials and Equipment Not Provided Item Source Microplate shaker Labline model 4625 or equivalent with 3 mm orbit Shaking incubator with microplate adaptor E amp K Scientific Vortemp 56 P N S 2056 or equivalent Luminex or Luminex based instrument MiraiBio Bio Rad or other Luminex instrument provider Materials Item Source Reagent Reservoirs 25 mL and 100 mL capacities Procarta Transcription Factor Nuclear Extraction Kit or Whole Cell Lysis Kit Di
16. m overexposure to light Do not use the vacuum over 10 seconds in any of the steps Store bead solution and the assay plate in the dark Header is clogged Follow the instructions in the Luminex instrument user documentation Procarta Transcription Factor Assay Kit User Manual Page 19 Contacting Panomics Observation Possible Cause Recommended Action Low signal or sensitivity High backgrounds Low protein concentration Increase the protein by at least 2 fold and retest Samples were not kept on ice or Binding Buffer is not cold Keep samples and Binding Buffer on ice Beads or reagents expired Verify the expiration date of the kit Beads stuck to the bottom of the plate Incubation too long during elution of TF bound Detection Probes Make sure the plate is agitated at 300 500 rpm for the recommended time and for at least 5 minutes before reading Do not exceed the 30 minute incubation time Reduced number of wash steps during elution of TF bound Detection Probes Do not reduce the number of wash steps to less than 5 Low or no fold induction observed Protein concentration too low or too high Optimize sample input Signals from instrument are saturated Reduce protein concentration of sample input Target protein not activated induced Review induction procedures You may need to change cell lines inducer or i
17. mmended Solutions Filter plate leakage Vacuum pressure too high Adjust the vacuum pressure to 2 3 mm Hg as recommended in Denaturing the Eluted Detection Probes on page 15 Filter Plate is misaligned at an angle during incubation processing Set the Filter Plate Utility Plate assembly on a flat level surface during incubation processing Leakage from capillary action After each vacuum step blot the bottom of the Filter Plate using paper towels or absorbent paper Plate Seal applied with too much force Do not use a rubber roller to seal the plate High CV Low bead count Sample not prepared properly Make sure the samples are eluted properly by using an accurate counterweight when centrifuging Bottom of the Filter Plate is not dry Contamination from re using the Plate Seal After each vacuum step blot the bottom of the Filter Plate using paper towels or absorbent paper Use a new Plate Seal for each incubation step Contamination from Wash Buffer Volume of bead solution is incorrect Be careful not to splash Wash Buffer during wash steps into adjacent wells Make sure the volume of Capture Beads is correct Beads are clumping Vacuum pressure too high Vortex the bead solution well before using in the assay Use 2 3 mm Hg vacuum pressure Filter ruptured due to excess vacuum time Dyes contained in the beads are photo bleached fro
18. n from a whole cell lysate or 250 ng of protein from a nuclear extract The xMAP technology developed by Luminex Corp combines flow cytometry fluorescent dyed microspheres beads lasers and digital signal processing to effectively allow multiplexing of up to 100 unique assays within a single sample Assay Overview Cis I ye Incubati ALL S ncubation Cis o tep 1 Cis Cis v TF Ur Cis TF y Separation Step 2 Y Cis HS Step 3 Cis Ci P Qi Az Luminex Assay bead bead V otep 4 O ms 9 ww y Step 5 Assay overview Step Action 1 Incubate nuclear extract or cell lysate with biotin labeled DNA binding probes to form protein DNA complexes 2 Separate protein DNA complexes from unbound probes with a separation plate Page 6 Procarta Transcription Factor Assay Kit User Manual Procarta Transcription Factor Assay Kit Contents and Handling Conditions Assay overview continued Step Action 3 Elute TF bound detection probes from the separation plate 4 Denature and hybridize eluted detection probes with TF specific antisense conjugated beads 5 Detect Streptavidin conjugated R phycoerythrin SAPE probe bound beads with a Luminex instrument Available Kit Procarta TF Assay Kits are available in 14 and 1 plate 96 well formats for the Formats standard 40 plex Assay kits contain all the reagents required to detect transcription factors from prepared whole cell ly
19. nduction conditions Contacting Panomics Technical Help 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 techsupport_europe panomics com Europe or visit our website www panomics com for an updated list of FAQs and product support literature For Additional For information about Panomics products or for ordering information contact your Services Regional Sales Manager or visit our website at www panomics com Page 20 Procarta Iranscription Factor Assay Kit User Manual Appendix Appendix Bead Analyte The following tables provide the bead analyte associations for setting your Luminex Associations instrument Refer to your product insert for analytes included in your kit Bead Analyte Bead Analyte 7 Elk1 42 PPAR 11 NF B 43 omad 12 FAST1 44 RUNX AML 17 Oct 45 Brn3 18 p53 46 CEBP 19 Pax3 47 NF Y 20 NF E2 51 c myb 21 AP2 52 CREB 25 NF E1 YY1 53 ER 26 ATF2 54 GR PR 27 NF 1 55 HIF 1 28 ISRE 56 FKHR 29 Pax5 61 GATA 32 Nkx 2 5 62 IRF 33 AR 63 Stat 34 ETS PEA 64 HNF1 35 AP1 65 STAT4 36 E2F1 66 STAT5 37 MyoD 73 MEF2 41 STAT3 76 NFAT Procarta Transcription Factor Assay Kit User Manual Page 21 Appendix Il Appendix Il Sample and Blank Plate Layouts 1 2 3 4 5 6 7 8 9 10 11 12 A PCa NEP Sample A 2 ug from NE treat
20. paring samples such that the addition of 10 uL of sample will contain a total of 4 0 2 0 1 0 and 0 5 ug of protein Assay Background Control Assay background is the assay signal median fluorescence intensity MFI generated by the assay components in the absence of sample We recommend you run an assay background control in every experiment and subtract the assay background MFI from each sample MFI when analyzing the data Whole Cell Lysate and Nuclear Extract Controls Whole cell lysate and nuclear extract controls enable you to monitor assay performance These controls are provided in the kit and are also available separately We recommend using at least one set of positive and negative controls in every experiment to monitor assay performance Technical Replicates These are replicates from a single extract or lysate sample Biological Replicates These are replicates from different extracts or lysates but the extracts or lysates are biologically equivalent Procarta Transcription Factor Assay Kit User Manual Guidelines for Assay Design and Data Analysis Replicate Recommendations For assays with 3 6 biological replicates run 1 assay well biological sample For assays without biological replicates run 3 technical replicate assay wells biological sample Analyzing Data Assay Precision Run assays of the same sample in triplicate Calculate standard deviation and assay coefficient of variation 96CV std dev mean
21. res adequate plate sealing while avoiding any leakage due to capillary action IMPORTANT To avoid Filter Plate leakages do not seal Filter Plates using a rubber roller or equivalent as they apply significant pressure resulting in leakage Seal all unused wells with the provided Plate Seal to ensure proper vacuum pressure To set up and calibrate the manifold Step Action 1 Set up the Filter Plate vacuum manifold as shown below Follow the manufacturer s manual for details Filter Pump Protection MultiScreen gt um acuum Vacuum Flask Manifold Trap In House Vacuum Calibrate the vacuum pressure using the Utility Plate a Place the Utility Plate on top of the manifold Turn on the vacuum Press the corners of the Utility Plate to form a tight seal o oc Set the pressure to 2 3 mm of Hg IMPORTANT During filtration maintain the vacuum between 2 3 mm Hg Higher vacuum may result in the loss of Capture Beads Procarta Iranscription Factor Assay Kit User Manual Assay Procedure Operating the To operate the manifold Manifold Step Action 1 Once the vacuum is set correctly remove the Utility Plate Check vacuum calibration periodically As a general guideline 200 uL of solution should take approximately 2 5 seconds to clear the well of a Filter Plate 2 For all filtration steps turn the Filter Plate vacuum manifold on transfer the Filter Plate to the vacuum manifold
22. reservoirs and multi channel pipets If you are running several partial plates you may Probes not have sufficient reagents for all steps For 1 4 plate kits there is not sufficient reagents to process the samples using reagent reservoirs and multi channel pipets For 1 4 plate kits perform reagent additions using a single channel pipet Before You Start Turn on the Luminex based reader at least 30 minutes before you intend to read your plate To denature the eluted Detection Probes Step Action 1 Seal the samples wells in the Sample Collection Plate with a PCR Plate Seal Using another PCR Plate Seal cover any unused wells IMPORTANT Do not use a foil Plate Seal as it will stick to the plate at high temperatures Denature samples at 95 C for 5 minutes using a PCR instrument or heat block Place samples on ice for 5 minutes Place the Filter Plate on top of the Utility Plate Cut a foil Plate Seal to size and cover any unused wells on the Filter Plate Pre wet the Filter Plate by dispensing 150 uL of Wash Buffer into each well and incubating the Filter Plate at room temperature for 5 minutes Remove the Wash Buffer using the vacuum manifold When all Wash Buffer has been filtered remove the Filter Plate from the vacuum manifold and blot the bottom of the Filter Plate with paper towels and place the Filter Plate on top of the Utility Plate Procarta Transcription Factor Assay Kit User M
23. sates or nuclear extracts including nuclear extract and whole cell lysate control samples Kits for the preparation of whole cell lysates or nuclear extracts are sold separately Additional Procarta TF Assay Kit are available as User selected 3 39 plex assay kits in 1 plate formats Selected TFs are provided in premixed ready to use format All assay kits include NF B in order to run the positive and negative controls provided in the kit Procarta Transcription Factor Assay Kit Contents and Handling Conditions Kit Contents and The Procarta TF Assay Kit contains the following components Refer to the product Storage insert for quantities and details of components supplied Procarta Transcription Factor Assay Kit components Component Description Storage Positive Control Nuclear Untreated HeLa nuclear extract with addition of 80 C Extract NF B recombinant protein Negative Control Nuclear Untreated HeLa nuclear extract 80 C Extract Positive Control Whole Untreated HeLa Whole Cell Lysate with 80 C Cell Lysate addition of NF B Recombinant Protein Negative Control Whole Untreated HeLa Whole Cell Lysate 80 C Cell Lysate Detection Probes Biotin labeled DNA binding consensus 20 C premixed sequences for specific TFs Sample Dilution Buffer Aqueous buffered solution for dilution of 20 C nuclear extract or whole cell lysate samples DIT 100 mM Aqueous solution for stabilizing protein 20
24. say design and data analysis Sample and assay preparation Set up and operation of the vacuum manifold system Assay procedure gt 9 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 Not for diagnosis of disease in humans or animals For More For information about the Procarta products mentioned in this manual visit our Information website at www panomics com Procarta Transcription Factor Assay Kit User Manual Page 5 About the Procarta Transcription Factor Assay Kit About the Procarta Transcription Factor Assay Kit Fundamentals of The Procarta TF assays combine two technologies a patent pending detection Procarta probe separation method and xMAP multi analyte profiling beads Together these Transcription two technologies enable quantitative measurement of DNA binding activity of up to Factor Assay 40 transcription factors from as little as 500 ng of protei
25. versified Biotech P N RESE 3000 RESE 1000 Panomics P N PC5101 P N PC5102 Protein Determination Kit Bio Rad DC Protein Assay Kit P N 500 01 12 or equivalent Aluminum Foil MLS Procarta Transcription Factor Assay Kit User Manual Page 9 Guidelines for Assay Design and Data Analysis Guidelines for Assay Design and Data Analysis Overview Preparing Samples Optimizing Sample Input Running Assay Page 10 Controls Running Replicates Here we provide information and guidelines on the following Preparing samples Optimizing sample input Running assay controls Running replicate samples SS 9 9 Analyzing data Note An example experimental plate layout is provided in Appendix II on page 22 Protein concentration of sample inputs should be in the range of 400 2000 ug mL As a starting point we recommend that each 10 uL sample contain a total of 2 ug of protein For example dilute samples to 0 2 ug mL using Working Sample Dilution Buffer and then load 10 pL We recommend running a 4 point 2 fold serial dilution of the sample diluting with Working Sample Dilution Buffer to ensure you are operating in the linear range of the reader and assay For nuclear extracts initially we recommend preparing samples such that the addition of 10 uL of sample will contain a total of 2 0 1 0 0 5 and 0 25 ug of protein For whole cell lysates initially we recommend pre
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