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Signosis, Inc. Innovative Plate Assay Solutions TF Activation Profiling Plate Array II Catalog Number: FA-1002 (For Research Use Only) Introduction Materials Provided with the Kit Transcription factors (TFs) are a group of cellular proteins that play essential roles in regulating gene Component Qty Store at expression. They act as sensors to monitor cellular 96-Well Plates (with 2 RT changes and convert signals into gene expression. aluminum adhesive seal) Often, a specific cellular signal pathway can activate Isolation Columns 2 RT multiple TFs. The expression of a specific gene can Elution Buffer 400µL RT also be under the control of multiple TFs. Thus, TF Plate Hybridization Buffer 20mL RT monitoring the activation of multiple TFs 5X Plate Hybridization Wash 60mL RT simultaneously is critical to understanding the Buffer molecular mechanism of cellular regulation underlying 5X Detection Wash Buffer 60mL RT cell signaling and gene expression. Signosis, Inc.’s TF Blocking Buffer 60mL RT Activation Profiling Plate Array II is used for Filter Wash Buffer 5mL 4°C monitoring 96 different TFs simultaneously from one Filter Binding Buffer 1mL 4°C sample. Substrate A 2mL 4°C Substrate B 2mL 4°C Principle of the assay Streptavidin-HRP Conjugate 40µL 4°C Substrate Dilution Buffer 16mL 4°C Signosis, Inc.’s TF Activation Profiling Plate Array II TF Binding Buffer Mix 60µL -20°C is used for monitoring the activation of multiple TFs TF Probe Mix II 20µL -20°C simultaneously. With this technology a series of biotin-labeled probes are made based on the consensus sequences of TF DNA-binding sites. When the probe Before Starting the Experiment mix incubates with nuclear extracts, individual probes Prepare the Following: will find its corresponding TF and form TF-probe complexes, which can be easily separated from free 1. Place Filter Binding Buffer and Filter Wash probes through a spin-column purification. The bound Buffer on ice so they are chilled for the assay (for probes are detached from the complex and analyzed at least 10 minutes). through hybridization with the 96-Well Plate. Each 2. Warm up TF Plate Hybridization Buffer and well is specifically pre-coated with complementary Hybridization Wash Buffer to 42˚C before use. sequences of the probes. The captured DNA probe is 3. Aliquot 500µL of ddH2O in a 1.5mL further detected with Streptavidin-HRP Conjugate. microcentrifuge tube per sample on ice so that it Luminescence is reported as relative light units (RLUs) is chilled for the assay (for at least 10 minutes). on a microplate luminometer 4. Dilute 60mL of 5X Plate Hybridization Wash Buffer with 240mL of ddH2O before use. Materials Required but Not Provided 5. Dilute 60mL of 5X Detection Wash Buffer with Nuclear Extraction Kit from Signosis (SK-0001) 240mL of ddH2O before use. PCR machine and PCR tubes 6. Dilute 40µL Streptavidin-HRP in 20mL Microcentrifuge working at 4 °C Blocking Buffer (1:500 dilution). Hybridization incubator at 42°C Plate-Shaker Please Read the Plate reader for luminescent detection ddH2O (DNAase-free) Assay Procedure 8 and 12 Multi-channel pipettes Before You Begin www.signosisinc.com [email protected] [email protected] 1(408)-747-0771 Home Page Questions / Comments Technical Support Telephone Assay Procedure TF/ DNA Complex Formation 1. Mix the following components for each reaction in a tube 15µL TF Binding Buffer Mix 5µL TF Probe Mix II XµL Nuclear Extract (5μg-15μg recommended) XµL ddH2O (add up to final volume) 30µL Reaction Mix [final volume] 2. Incubate the Reaction Mix at room temperature (20-23oC) for 30 minutes. Separation of TF DNA Complex from Free Probes 3. Equilibrate an Isolation Column by adding 200µL pre-chilled Filter Binding Buffer. Centrifuge the column with the collection tube at 6,000rpm for 1 minute in a microcentrifuge at room temperature. 4. Transfer the 30µL Reaction Mix directly onto the filter in the center of the Isolation Column (avoiding bubbles). 5. Incubate on ice for 30 minutes. DO NOT incubate longer than 30 minutes; this will result in high background. sample, go to step 16. before proceeding to the 6. Add 500µL pre-chilled Filter Wash Buffer to the hybridization phase. Isolation Column and incubate for 3 minutes on 16. Skip this step if you did not freeze your ice. sample for future use. 7. Centrifuge the Isolation Column with the A) Thaw your sample back to an aqueous phase collection tube at 6,000 rpm for 1 minute in a at room temperature. microcentrifuge at 4oC. Discard the flow through B) Redistribute the sample into PCR tubes to be from the collection tube. reheated at 98°C for 5 minutes. 8. Wash the column by adding 500µL pre-chilled C) Afterwards, immediately place the PCR Filter Wash Buffer to the Isolation Column on ice. tubes on ice. 9. Centrifuge the Isolation Column with the D) You may now proceed to Step 17. collection tube for 1 minute at 6,000rpm in a microcentrifuge at 4oC. Hybridization of Eluted Probe with Hybridization 10. Repeat steps 8-9 for an additional 3 times for a Plate total a 4 washes. 17. Remove the clear adhesive film sealing from Elution of Bound Probe the provided 96-Well Plate. 18. Aliquot 10mL pre-warmed TF Plate 11. Add 100µL of Elution Buffer onto the center of Hybridization Buffer to a dispensing reservoir Isolation Column, and incubate at room (DNase free) and then add 600µL denatured temperature for 5 minutes. probes. Mix them together by gently shaking 12. Place the Isolation Column on a new 1.5mL the reservoir. microcentrifuge tube and centrifuge at 10,000 19. Using a 12 multi-channel pipette 100µL of rpm for 2 minutes at room temperature. the mixture from step 18. into the 13. If you have yet to do so, chill 500µL ddH2O corresponding wells with 8 multi-channel (DNAase free) in a 1.5mL microcentrifuge tube pipette immediately. on ice for at least 10 minutes, and keep on ice. 14. Transfer the eluted probe to a PCR tube and Note: If you wish to have a blank to denature the eluted probes at 98°C for 5 minutes. compare your wells against, select one TF 15. Immediately transfer the denatured probes to the you are not interested in and determine its chilled ddH2O from Step 13 and place on ice. location on the plate by using the diagram on The samples are ready for the hybridization phase the third page. Add 100µL TF Plate of the assay. You can store the sample at -20oC Hybridization Buffer only without the eluted for future use. If you decided to store your probe. www.signosisinc.com [email protected] [email protected] 1(408)-747-0771 Home Page Questions / Comments Technical Support Telephone 20. Firmly seal the wells with the aluminum 26. Add 95µL of diluted Streptavidin-HRP adhesive seal to secure well contents. Press Conjugate to each well by row with a 12 multi- the foil over the letters and numbers on the channel pipette and incubate for 45 minutes at plate to help orient well designations. room temperature on a plate-shaker with gentle Hybridize the well contents to the plate by shaking. placing the 96-Well Plate in an incubator set 27. After the 45 minutes have elapsed, forcibly at 42°C overnight. remove the 96-Well Plate contents in an appropriate container. Complete removal of Detection of Bound Probe liquid at each wash by firmly tapping the plate against clean paper towels. 21. Remove the aluminum adhesive seal from 28. Wash the 96-Well Plate by adding 200µL 1X the experimental wells with a razor blade. Detection Wash Buffer to each well by row with Keep the unused wells sealed. a 12 multi-channel pipette. Incubate the plate 22. Invert the 96-Well Plate over an appropriate for 5 minutes with gentle shaking on a plate- container and expel the contents forcibly. shaker at room temperature. 23. Wash the plate by adding 200µL of pre- 29. Repeat step 28. two more times. At the third and warmed 1X Plate Hybridization Wash Buffer final wash, invert plate on clean paper towels for to each well by row with a 12 multi- 1 minute to remove excessive liquid. channel pipette. Incubate the plate for 5 28. Freshly prepare the Substrate Solution in the minutes with gentle shaking at room following ratio: temperature on a plate-shaker. Completely 1 part Substrate A / 1 part Substrate B / 8 parts remove at end of 5 minutes by tapping the Substrate Dilution Buffer. plate against clean paper towels. For example, for the entire 96-Well Plate: 24. Repeat step 23. two more times for a total of 1mL Substrate A three washes. 1mL Substrate B 25. Add 200µL of Blocking Buffer to each well 8mL Substrate Dilution Buffer by row with a 12 multi-channel pipette and 10mL Substrate Solution incubate for 5 minutes at room temperature 29. Add 95µL Substrate Solution to each well by with gentle shaking on a plate-shaker. row with a 12 multi-channel pipette and 26. Invert the plate over an appropriate incubate the solution in the wells for 1 minute at container to forcibly remove Blocking Buffer room temperature. from the wells. 30. Place the plate in the luminometer. Allow plate 25. If you have yet to do so: add 40µL of to sit inside machine for 4 minutes before Streptavidin-HRP Conjugate in 20mL reading. Set integration time to 1 second with no Blocking Buffer (1:500 dilution), enough for filter position. For the best results, read the plate the whole plate (6 sections).
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