EpiQuik™ BRD2 Binding Activity/Inhibition Assay Kit
Contents
1. EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric Base Catalog P 4058 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric is a complete set of essential components which enables an experimenter to colorimetrically quantify BRD2 binding activity inhibition The EpiQuik BRD2 Binding Activity Inhibition Assay Kit can be used with purified BRD2 proteins or nuclear extracts from fresh tissue or cultured cells from human and mouse Nuclear extracts can be prepared using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use Purified BRD2 can be isolated from recombinant proteins or from cell tissues Starting Materials Input materials can be nuclear extracts or purified BRD2 protein The amount of nuclear extracts for each assay can be 1 ug to 20 ug with an optimized range of 5 10 ug The amount of purified protein can be 10 ng to 500 ng depending on the purity of the protein Internal Control A BRD2 protein is provided in this kit as a control for the quantification of BRD2 binding activity Because BRD2 binding activity can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is val2. Incorrect absorbance reading Check if appropriate absorbance wavelength 450 nm is used Kit was not stored or handled properly Ensure all components of the kit were stored at the appropriate temperature and the cap is tightly capped after each opening or use No signal or weak signal in only the standard curve wells The standard amount is insufficiently added to the well in Step 2g Ensure a sufficient amount of standard is added The standard is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of BRD2 Control High background present in the blank wells Insufficient washing of wells Check if washing recommendations at each step is performed according to the protocol Contaminated by sample or standard Ensure the well is not contaminated from adding sample or standard accidentally or from using contaminated tips Incubation time with Diluted DA is too long The incubation time at Step 3d should not exceed 90 min Over development of color Decrease the development time in Step 4a before adding SS Stop Solution in Step 4b 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2014 09 23 P 405
3. Inhibition Assay Kit addresses these problems by using a unique procedure to measure BRD2 binding activity inhibition The kit has the following features e Fast procedure which can be finished within 6 hours e Innovative colorimetric assay which directly measures BRD2 binding activity in a 96 well plate using a standard microplate reade without the need for special reagents or expensive equipment e Both cell tissue extracts and purified BRD2 protein can be used which allows for the detection of inhibitory effects of BRD2 inhibitors in vivo and in vitro e High sensitivity and specificity BRD2 binding specific detection with a detection limit as low as 0 1 ng well bound BRD2 protein e Strip microplate format makes the assay flexible via manual or high throughput analysis e BRD2 control is included which allows the binding activity of BRD2 protein in the sample to be properly quantified e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE In an assay with this kit the unique BRD2 ligand is stably coated onto the strip well The sample is added into the well and BRD2 proteins contained in the sample bind to the ligand The bound BRD2 can be recognized with a BRD2 specific antibody and colorimetrically quantified through an ELISA like reaction The amount of bound BRD2 is proportional to the intensity of color development Start with nuclear extract or purified protein Incubate with ligand for 60 mi
4. also offers a nuclear extraction kit Cat OP 0002 which has been optimized for use with this kit see Related Products section Nuclear extracts should be stored at 80 C in aliquots until use 1 Working Buffer and Solution Preparation a Prepare Diluted WB 1X Wash Buffer 48 Assay Kit Add 13 ml of WB 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of WB 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted WB 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare 1X BRL BRD2 Ligand Solution Add 1 ul of BRL 100X BRD2 Ligand to 100 ul of BRS BRD2 Ligand Binding Solution 100 pl of 1X BRL will be required for each assay well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Piintedi 2014 0923 Epigentek Group Inc All rights reserved Products are for research use only P 4058 j bb co sa g see A c Prepare Diluted CA Capture Antibody Solution Dilute CA Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of CA to 1000 ul of Diluted WB 50 ul of Diluted CA will be required for each assay well d Prepare Diluted DA Detection Antibody Solution Dilute DA Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 2000 i e add 1 pl of DA to 2000 ul of Diluted WB 50 ul of Diluted DA
5. will be required for each assay well e Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of Diluted WB About 50 ul of this Diluted ES will be required for each assay well f Prepare Diluted BRD2 Control Standard Suggested Standard Curve Preparation First dilute BRD2 control with AB to a concentration of 40 ng ul by adding 2 ul of BRD2 control to 8 ul of AB Then further prepare concentration points of 2 4 10 20 and 40 ng ul according to the following chart Resultin Tube BRDZ O gp BD Concentration 1 0 5 ul 9 5 ul 2 ng ul 2 0 5 ul 4 5 ul 4 ng ul 3 1 0 ul 3 0 ul 10 ng ul 4 2 0 ul 2 0 ul 20 ng ul 5 4 0 ul 0 0 ul 40 ng ul Note Keep each of the diluted solutions except WB 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day The lower concentration point ex 0 5 ng ul can be also added if needed 2 BRD2 Binding Inhibition Reaction a Predetermine the number of strip wells required for your experiment It is advised to run replicate samples include blank and positive controls to ensure that the signal generated is validated Carefully remove un needed strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Add 100 ul of 1X BRL to each well Ensure the solution coa
6. 8 No signal or weak signal only in sample wells Protein sample is not properly extracted or purified Ensure your protocol is suitable for histone protein extraction For the best results it is advised to use Epigentek s Nuclear extraction Kit Cat No OP 0002 Sample amount added into the wells is insufficient Ensure a sufficient amount of nuclear extracts is used as indicated in Step 2 The sample can be titrated to determine the optimal amount to use in the assay Sample was not stored properly or has been stored for too long Ensure sample is stored in aliquots at 80 C with no more than 6 months nuclear extracts Little or no BRD2 in the sample This problem may be a result of many factors If the affecting factors cannot be determined use new or re prepared nuclear extracts Uneven color development Insufficient washing of the wells Ensure the wells are washed according to the guidance of washing and residue washing buffer is removed as much as possible Delayed color development or delayed stopping of color development in the wells Ensure color development solution or stop solution is added sequentially and is consistent with the order you added the other reagents e g from well A to well G or from well 1 to well 12 RELATED PRODUCTS Nuclear Extract Preparation OP 0002 EpiQuik Nuclear Extraction Kit Histone Deacetylase Activity Inhib
7. erse family of evolutionarily conserved protein interaction modules are found in histone acetyl transferases and other chromatin associated proteins More than 60 BRDs have been identified which in turn cluster into eight families based on structure sequence similarity BRDs bind selectively to acetylated lysines acting as readers of the histone code and have recently been shown to regulate transcription activity they also contain a druggable binding pocket Proteins that contain BRD2 have been implicated in the development of a large variety of diseases and inhibitors that target BRDs of the BET Bromodomains and extra terminal which inhibit BRD mediated protein protein interaction and have the potential to modulate multiple diseases including inflammation and cancer There are currently no conveniet methods used for detecting BRD2 binding activity inhibition Bead based prepcipitation requires electrophoresis and immunoblotting processes which make the assay inconvenient time consuming and low throughput The FRET based methods such as TR FRET and 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 AlphaLlSA allow for high throughput assays but require expensive equipment and purified proteins The EpiQuik BRD2 Binding Activity
8. idated Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 4058 48 Cat P 4058 96 Upon Receipt WB 10X Wash Buffer 12 ml 25 ml 47 BRL BRD2 Ligand 100X 50 ul 100 ul 20 C BRS Ligand Binding Solution 5ml 10 ml 47 BB Blocking Buffer 10 ml 20 ml 47 AB Assay Buffer 3 ml 6 ml 47 CA Capture Antibody 1000 ug mIl 5 ul 8 ul 4 DA Detection Antibody 200 g ml 6 ul 10 pl 20 C ES Enhancer solution 6 ul 10 ul 20 C DS Developing Solution 6 ml 12 ml 47 SS Stop Solution 6 ml 11 ml RT BRD2 Control 200 yg ml 8 ul 16 ul 20 C BRD2 Inhibitor 100 uM 20 ul 40 ul 20 C 8 Well Assay Strips With Frame 6 12 47 User Guide 1 1 RT For maximum recovery of the products centrifuge the original vial after thawing prior to opening the cap SHIPPING amp STORAGE The kit is sh
9. ipped in two parts one part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store BRL DA ES BRD2 Control and BRD2 Inhibitor at 20 C away from light 2 Store WB BRS BB AB CA DS and the 8 Well Assay Strips at 4 C away from light 3 Store all other components at room temperature The kit is stable for up to 6 months from the shipment date when stored properly Note 1 Check if wash buffer WB contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved 2 check if a blue color is present in DS Developing Solution If it is blue this indicates contamination of the solution and it should not be used To avoid contamination transfer the amount of DS required into a secondary container tube or vial before adding DS into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette or multiple channel pipette O Multiple channel pipette reservoirs O Aerosol resistant pipette tips 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2014 09 23 P 4058 Microplate reader capable of reading absorbance at 450 nm 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Distilled water Nuclea
10. ition Assay P 4034 P 4035 P 4036 P 4037 P 4003 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Epigenase HDAC Activity Inhibition Direct Assay Kit Epigenase HDAC Activity Inhibition Direct Assay Kit Fluorometric Epigenase Universal SIRTActivity Inhibition Assay Kit Epigenase Universal SIRT Activity Inhibition Assay Kit Fluorometric EpiQuik HAT Activity Inhibition Assay Kit Page 11 Printed 2014 09 23 P 4058
11. late reader within 2 to 10 min at 450 nm with an optional reference wavelength of 655 nm Note 1 Most microplate readers have the capability to carry out dual wavelength analysis and will automatically subtract reference wavelength absorbance from the test wavelength absorbance If your plate reader does not have this capability the plate can be read twice once at 450 nm and once at 655 nm Then manually subtract the 655 nm ODs from 450 nm ODs 2 If the strip well microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 BRD2 Calculation a Calculate the average replicate readings for the sample wells and blank wells b Calculate BRD2 binding activity or inhibition using the following formulas For simple calculation Sample OD Blank OD BRD2 Binding Activity OD min mg x 1000 Protein Amount ug x min Protein amount ug added into the reaction at step 2h Incubation time minutes at step 2j Example calculation Average OD450 of sample is 0 35 Average OD450 of blank is 0 05 Protein amount is 5 pg Incubation time is 60 minutes 1 hour 0 35 0 05 BRD2 Binding activity X 1000 1 OD min mg 5 x 60 For accurate binding activity calculation 1 Generate a standard curve and plot OD value versus amount of BRD2 Control Standard at each concentration point 2 Determine the slope as OD ng you can use Microsoft Excel statistical functions for s
12. lope calculation then calculate the amount of bound BRD2 using the following formulas 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 23 P 4058 Sample OD Blank OD BRD2 ng Slope BRD ng BRD2 Binding Activity ng min mg x 1000 Protein Amount ug X min Incubation time minutes at Step 2j For inhibition calculation Inhibitor Sample OD Blank OD Inhibition 1 X 100 No Inhibitor Sample OD Blank OD SUGGESTED BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1 well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted WB 2 5 ml 20 ml 40 ml 120 ml 240 ml BRL 1 ul 8 ul 16 ul 48 ul 96 ul BRS 100 ul 800 ul 1600 ul 4800 ul 9600 ul AB 50 ul 400 ul 800 ul 2400 ul 4800 ul BB 0 15 ml 1 2 ml 2 5 ml 7 5 ml 14 5 ml BRD2 control N A N A 2 uL optional 4 ul 4ul BRD2 Inhibitor N A N A 5 uL 10 uL 20 uL Diluted CA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted ES 50 ul 400 ul 800 ul 2400 ul 4800 ul DS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml ss 0 1 ml 0 8 ml 1 6 ml 4 8
13. mended to use 2 ug to 10 ug of nuclear extract per well or 20 ng to 100 ng of purified protein per well 3 The concentration of inhibitor to be added into the sample wells can be varied ex 1 uM to 1000 uM However the final concentration of the inhibitors before adding to the wells should be prepared with AB at a 1 10 ratio ex add 0 5 ul of inhibitor to 4 5 ul of AB so that the original solvent of the inhibitor can be reduced to 1 of the reaction solution or less The BRD inhibitor JQ1included in the kit can be used as the control inhibitor Tightly cover strip plate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 60 120 min Note 1 The incubation time may depend on intrinsic BRD2 protein binding activity However in general 60 90 min incubation is suitable for active purified proteins and 90 120 min incubation is suitable for nuclear extracts 2 The Adhesive Covering Film can be cut to the required size to cover the strips based on the number of strips to be used Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time 3 Antibody Binding amp Signal Enhancing a Add 50 ul of the Diluted CA to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 60 min b Remove
14. ml 9 6 ml SUGGESTED STRIP WELL SETUP Table 2 The suggested strip well plate setup for BRD2 assay in a 48 assay format in a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 9 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B BRD2 2 ng BRD2 2 ng Sample Sample Sample Sample 03 BRD2 4 ng BRD2 4 ng Sample Sample Sample Sample D BRD2 10 ng BRD2 10 ng Sample Sample Sample Sample E BRD2 20 ng BRD2 20 ng Sample Sample Sample Sample F BRD2 40 ng BRD2 40 ng Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Cause Suggestion No signal or weak signal in both the positive control and sample wells Reagents are added incorrectly Check if reagents are added in the proper order with the right amount and if any steps in the protocol may have been omitted by mistake Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly
15. nutes NS NNN Wash wells then add Ap affinity antibody Wash wells then add detection antibody and enhancer solution Add color developing solution for color develop ment then measure absorbance Schematic procedure of the EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 0D450 nm 0 3 0 25 l 0 2 i 2 015 J 0 1 4 l 0 05 z i 0 T T T T T T T 1 0 7 7 7 1 0 2 5 10 0 45 0 4 5 0 35 5 BRD2 Binding Activity OD hour 0 5 10 15 20 25 30 35 40 BRD2 Control ng Nuclear extracts ug Nuclear extracts were prepared from HeLa cells and the Illustrated standard curve generated with BRD2 control ODs generated from BRD2 are measured ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of nuclear extracts for each assay can be 1 ug to 20 ug with an optimized range of 5 10 ug The amount of purified protein can be 10 ng to 500 ng depending on the purity of the protein Nuclear Extraction You can use your own successful method for preparing nuclear extracts from treated or untreated samples Epigentek
16. r extracts or purified BRD2 protein Oo O 0 0 2 O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic applications Intellectual Property The EpiQuik BRD2 Binding Activity Inhibition Assay Kit Colorimetric and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Bromodomains BRDs which are a div
17. the Diluted CA solution from each well c Wash each well three times with 150 ul of the Diluted WB each time d Add 50 ul of the Diluted DA to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min e Remove the Diluted DA solution from each well f Wash each well four times with 150 ul of the Diluted WB each time g Add 50 ul of the Diluted ES to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 h Remove the Diluted ES solution from each well Wash each well five times with 150 ul of the Diluted WB each time Note Ensure any residual wash buffer in the wells is removed as much as possible at each wash step 4 Signal Detection a Add 100 ul of DS to each well and incubate at room temperature for 1 to 10 min away from light Begin monitoring color change in the sample wells and control wells The DS solution will turn blue in the presence of sufficient BRD2 protein Add 100 ul of SS to each well to stop enzyme reaction when color in the positive control wells turns medium blue The color will change to yellow after adding SS and the absorbance should be read ona microp
18. ts the bottom of the well evenly c Cover strip plate with plate seal or Parafilm M and incubate at 37 C for 120 min d Remove the 1X BRL from each well Add 150 ul of BB Blocking Buffer to each well then cover with Parafilm M or aluminum foil and incubate at 37 C for 30 min e Wash each well three times with 150 ul of the Diluted WB 1X Wash Buffer each time 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 23 Epigentek Group Inc All rights reserved Products are for research use only P 4058 Blank Wells Add 100 ul of AB to each blank well Standard Wells Add 49 ul of AB and 1 ul of Diluted BRD2 Control Standard to each standard well to a minimum of five wells each at different concentrations between 1 and 20 ng ul based on the dilution chart in Step 1f see Table 2 in the Suggested Strip Well Setup section as an example Sample Wells Without Inhibitor Add 45 to 48 ul of AB 2 to 5 ul of your nuclear extracts or 2 to 5 ul of your purified BRD2 protein Total volume should be 50 ul per well Sample Wells With Inhibitor Add 40 to 43 ul of AB 2 to 5 ul of your nuclear extracts or 2 to 5 ul of purified BRD2 protein and 5 ul of inhibitor solution Total volume should be 50 ul per well Note 1 Follow the diagrams under the Suggested Working Buffer amp Solution Setup section 2 It is recom
Download Pdf Manuals
Related Search