EpiQuik™ Global Di-Methyl Histone H4R3
Contents
1. EpiQuik Global Di Methyl Histone H4R3 Quantification Kit Colorimetric Base Catalog P 3090 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Global Di Methyl Histone H4R3 Quantification Kit Colorimetric is suitable for specifically measuring global histone H4 arginine 3 di methylation from a broad range of species such as mammals plants fungi and bacteria in a variety of forms including cultured cells and fresh tissues Histone extracts can be prepared by using your own successful method For your convenience and the best results Epigentek offers a histone extraction kit Cat OP 0006 optimized for use with this kit Histone extracts can be used immediately or stored at 80 C for future use Input Material Input materials can be histone extracts or nuclear extracts The amount of histone extracts for each assay can be 0 1 ug to 2 ug with an optimal range of 0 5 to 1 yg Internal Control The assay control methylated histone H4 Arg 3 is provided in this kit for the quantification of global di methyl histone H4R3 Because content of di methyl histone H4R3 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 validated 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 Wear2. 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 2015 06 23 Epigentek Group Inc All rights reserved Products are for research use only P 3090 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3090 48 Cat P 3090 96 Upon Receipt WB 10X Wash Buffer 14 ml 28 ml 47 HB Histone Buffer 4ml 8 ml 47 BB Blocking Buffer 10 ml 20 ml 47 MER3 Capture Antibody 1000X 5 ul 10 pl 4 RDA Detection Antibody 2000X 6 ul 12 ul 20 C ES Enhancer solution 6 ul 12 ul 20 C DS Developer Solution 5 ml 10 ml 47 SS Stop Solution 5 ml 10 ml RT Di Methyl H4R3 control 50 ug ml 10 ul 20 ul 20 C 8 Well Assay Strips With Frame 6 12 47 Adhesive Covering Film 1 1 RT User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in three parts the first part at ambient room temperature and the second and third parts on frozen ice packs at 4 C Upon receipt 1 Store RDA ES and Di Methyl H4R3 control at 20 C away from light 2 Store WB HB BB MER3 DS and 8 Well Assay Strips at 4 C away from light and 3 Store remaining components SS and Adhesive Covering Film at room
3. factors If the affecting factors cannot be determined use new or re prepared histone 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 12 110 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 2015 06 23 P 3090 RELATED PRODUCTS Histone Extract Preparation OP 0006 EpiQuik Total Histone Extraction Kit PRMT methyltransferase Activity Inhibition Assy P 3088 Epigenase PRMT Methyltransferase Type Il Specific Activity Inhibition Assay Kit Colorimetric 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 11 Printed 2015 06 23 P 3090
4. temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note 1 Check if WB 10X Wash Buffer contains salt precipitates before use If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved and 2 check if a blue color present in DS Developer Solution which would indicate contamination of the solution and 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 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 O Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm 1 5 ml microcentrifuge tubes Oo OF O Q OQ Incubator for 37 C incubation Page 2 Printed 2015 06 23 P 3090 O Distilled water O Histone extracts O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik Global Di Methyl Histone H4R3 Quantification Kit Colorimetric is tested against predetermined specifications to ensure consistent product quality Epigentek guara
5. Histone Extraction You can use your method of choice for preparing histone extracts from the treated and untreated samples Epigentek also offers a histone extraction kit Cat OP 0006 optimized for use with this kit Histone extracts should be stored in aliquots at 80 C 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 Diluted MER3 Capture Antibody Solution Dilute MER3 Capture Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of MER3 to 1000 ul of Diluted WB 50 ul of Diluted MER3 will be required for each assay well c Prepare Diluted RDA Detection Antibody Solution Dilute RDA Detection Antibody with Diluted WB 1X Wash Buffer at a ratio of 1 2000 i e add 1 ul of RDA to 2000 ul of Diluted WB 50 ul of Diluted RDA will be required for each assay well d Prepare Diluted ES Enhancer Solution Dilute ES Enhancer Solution with Diluted WB 1X Wash Buffer at a ratio of 1 5000 i e add 1 ul of Wash Buffer at a ratio of 1 5000 i e add 1 ul of ES to 5000 ul of WB About 50 ul of this Diluted ES will be required for each assay well e Prepare Diluted di methyl H4R3 Control Standa
6. eserved Products are for research use only Printed 2015 06 23 P 3090 125 10 Start with histone extracts Di Methyl H4R3 binds to the assay wells A Wash wells then add capture antibody 0 T T T 1 0 0 5 1 1 5 2 Histone extracts ug Di methy H4 R3 ng o Wash wells then add detection antibody and Histone extracts were prepared from MDA 231 cells using the enhancer solution EpiQuik Total Histone Extraction Kit and the amount of dimethyl H4R3 was measured using the EpiQuik Global Dimethyl Histone H4R3 Quantification Kit Colorimetric Add color developing solution for color develop ment then measure absorbance Schematic procedure of the EpiQuik Global Di Methyl Histone H4R3 Quantification Kit Colorimetric OD450 nm 0 2 4 6 8 10 Di methyl H4R3 control ng Illustrated standard curve generated with di methyl H4R3 control PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of histone extracts for each assay can be between 0 1 ug and 2 ug with an optimal range of 0 2 to 0 5 ug 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 Printed 2015 06 23 Epigentek Group Inc All rights reserved Products are for research use only P 3090 j bb co sa g see A
7. he standard is degraded due to improper storage conditions Follow the Shipping amp Storage guidance in this User Guide for storage of H4R3 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 RDA 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 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 histone extraction Kit Cat No OP 0006 Sample amount added into the wells is insufficient Ensure a sufficient amount of histone 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 histone extracts Little or no di methylH4 R3 in the sample This problem may be a result of many
8. i Methyl H4R3 ng mg protein _ x 1000 Slope x Protein Amount ug Histone extract added into sample wells at step 2d Page 8 Printed 2015 06 23 P 3090 SUGGESTED BUFFER AND SOLUTION SETUP Table 1 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagenis 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 HB 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 H4R3 control N A N A 4 uL optional 8ul 8 ul Diluted MER3 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted RDA 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted ES 50 ul 400 ul 800 ul 2400 ul 4800 ul Developer Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml Stop Solution 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml SUGGESTED STRIP WELL SETUP Table 2 The suggested strip well plate setup for dimethyl H4R3 quantification 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 Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B H4R3 0 5ng H4R3 0 5ng Sample Sample Sample Sample c H4R3 1 ng H4R3 1 ng Sample Sample Sample Sample D H4R3 2 ng H4R3 2
9. l of Diluted H4R3 control to each standard well with a minimum of five wells each at a different concentration between 0 5 and 10 ng ul based on the dilution chart in Step 1e see Table 2 under the Suggested Strip Well Setup section as an example Sample Wells Add 46 to 49 ul of HB and 1 to 4 ul of your histone extracts Total volume should be 50 ul per well Note 1 Follow the suggested well setup diagrams 2 It is recommended to use 0 2 ug to 0 5 ug of histone extract per well Tightly cover strip well microplate with Adhesive Covering Film to avoid evaporation and incubate at 37 C for 90 to 120 min Note 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 Add 150 ul of BB block buffer to each well then cover with Parafilm M or aluminium foil and incubate at 37 C for 30 min 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 and Signal Enhancing a Add 50 ul of the Diluted MER3 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted MER3 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 RDA to each well then cover with Parafilm M or aluminium foil and incubate at ro
10. lly 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 Di Methyl H4R3 Calculation 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 Calculate the average duplicate readings for the sample wells and blank wells Calculate histone H4R3 di methylation change using the following formula Treated Tested Sample OD Blank OD Di Methyl H4R3 R ee ee oa Untreated Control Sample OD Blank OD Example calculation Average OD450 of treated sample is 0 5 Average OD450 of untreated control is 0 9 Average OD450 of blank is 0 1 0 5 0 1 Di Methyl H4R3 __ x 1008 50 0 9 0 1 For accurate calculation 1 Generate a standard curve and plot OD value versus amount of H4R3 control at each concentration point 2 Determine the slope as OD ng you can use Microsoft Excel statistical functions for slope calculation then calculate the amount of di methyl H4R3 using the following formulas Sample OD Blank OD D
11. methionine SAM as a methyl donor and transfer it to the guanidinium side chain of arginine Based on the position of methyl group addition the PRMTs can be classified into type CARM1 PRMT1 PRMT2 PRMT3 PRMT6 and PRMT8 and type II PRMT5 and PRMT7 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 2015 06 23 Epigentek Group Inc All rights reserved Products are for research use only P 3090 NZ c AdoMet NH i ee AN A N 1 AdoMet Adol oO HN NH NH Z N z c c NH NH q Tas ar aS gt yok gt X y oy symmetric asymmetric Fig 1 Histone arginine methylation reaction catalyzed by PRMTs Symmetric di methylation of histone H4 arg3 H4R3 is catalyzed by type II PRMTs which are found to be strongly implicated in diseases like cancer For example PRMT5 plays a role in the repression of certain tumor suppressor genes such as RB tumor suppressors while PRMT7 overexpression is observed in breast cancer The global H4R3 di methylation can be changed by inhibition or activation of type II PRMTs Therefore quantitative detection of global symmetric di methyl histone H4R3 would provide useful information for better understanding epigenetic regulation of gene activation and silencing as well as for developing PRMT targeted drugs The EpiQuik Global Di Methyl Histone H4R3 Quantification Ki
12. ng Sample Sample Sample Sample E H4R3 5 ng H4R3 5 ng Sample Sample Sample Sample F H4R3 10 ng H4R3 10 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 Ensure the incubation time and temperature described in the protocol are followed correctly Incubation time and temperature are incorrect 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 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 9 Printed 2015 06 23 P 3090 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 2c Ensure a sufficient amount of standard is added T
13. ntees 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 Global Di Methyl Histone H4R3 Quantification Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application A BRIEF OVERVIEW Arginine histone methylation is one of the many important epigenetic marks and is essential for the regulation of multiple cellular processes Arginine methylation of histones H3 Arg2 8 17 26 and H4 Arg3 promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases PRMTs There are 9 types of PRMTs found in humans but only 7 members are reported to methylate histones They can mediate mono or dimethylation of arginine residues These enzymes use S adenosyl
14. om temperature for 30 min e Remove the Diluted RDA 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 carefully cover with Parafilm M or aluminium foil and incubate at room temperature for 30 min h Remove the Diluted ES solution from each well i Wash each well with 150 ul of the Diluted WB each time for five times 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 Web www epigentek com Printed 2015 06 23 Epigentek Group Inc All rights reserved Products are for research use only P 3090 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 methylated products 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 microplate 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 automatica
15. rd Suggested Standard Curve Preparation First dilute H4R3 Control with HB histone buffer to 10 ng ul by adding 2 ul of H4R3 Control to 8 ul of HB histone buffer Then further prepare five concentrations by combining the 10 ng yl Diluted H4R3 Control with HB into final concentrations of 0 5 1 2 5 and 10 ng ul according to the following dilution chart Tube ARa HB ERS 20 ng pl Concentration 1 1 0 ul 19 0 ul 0 5 ng ul 2 1 0 ul 9 0 ul 1 ng ul 3 1 0 ul 4 0 ul 2 ng ul 4 2 0 ul 2 0 ul 5 ng ul 5 4 0 ul 0 0 ul 10 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 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 2015 06 23 Epigentek Group Inc All rights reserved Products are for research use only P 3090 2 Histone Binding 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 Blank Wells Add 49 ul of HB to each blank well Standard Wells Add 49 ul of HB and 1 u
16. t Colorimetric is designed to quantitatively detect global di methyl histone H4R3 This kit has the following advantages e Quick and efficient procedure which can be finished within 3 5 hours e Innovative colorimetric assay without the need for radioactivity electrophoresis or chromatography e Specifically captures symmetric di methylated H4R3 with the detection limit as low as 0 5 ng well and detection range from 100 ng to 2 ug well of histone extracts e The control is conveniently included for the quantification of di methylated H4R3 e Strip microplate format makes the assay flexible manual or high throughput e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik Global Di Methyl Histone H4R3 Quantification Kit Colorimetric is designed for measuring global histone H4R3 di methylation In an assay with this kit the histone proteins are stably spotted on the strip wells The di methy histone H4R3 can be recognized with a high affinity antibody and detected with a detection antibody followed by a color development reagent The ratio of di methylated H4R3 is proportional to the intensity of absorbance The absolute amount of di methylated H4R3 can be quantitated by comparing to the standard control 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 Epigentek Group Inc All rights r
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