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1. EpiQuik Total Histone H4 Quantification Kit Colorimetric Base Catalog P 3072 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Total Histone H4 Quantification Kit Colorimetric is suitable for specifically measuring total histone H4 from mammals 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 50 ng to 1 ug with an optimal range of 0 1 to 0 2 ug Internal Control The assay control purified histone H4 is provided in this kit for the quantification of total histone H4 Because content of histone H4 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 Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Far
2. 1 ul of Enhancer Solution to 5000 ul of C1 About 50 ul of this Diluted Enhancer will be required for each assay well e Prepare Diluted Standard Control Suggested Standard Curve Preparation First dilute Standard Control with C2 Histone Binding Buffer to 50 ng ul by adding 5 ul of Standard Control to 5 ul of C2 Histone Binding Buffer Then further prepare five concentrations by combining the 50 ng ul Diluted Standard Control with C2 Histone Binding Buffer into final concentrations of 1 2 5 10 20 and 50 ng ul according to the following dilution chart 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 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072 We ge Sa e 1 1 0 ul 49 0 yl 1 ng l 2 1 0 ul 24 0 ul 2 ng ul 3 1 0 ul 9 0 ul 5 ng ul 4 1 0 ul 4 0 ul 10 ng ul 5 2 0 ul 3 0 ul 20 ng ul 6 3 0 ul 0 0 ul 50 ng pl Note Keep each of the diluted solutions except Diluted C1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted C1 should be discarded if not used within the same day 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 re
3. 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 Signal Reporter 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 Stop Solution in Step Ab 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 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
4. SC 50 ng SC 50 ng Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample 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 8 Printed 2014 09 22 P 3072 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 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 2c 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 Standard
5. for research use only Page 9 Printed 2014 09 22 P 3072 Uneven color Insufficient washing of the Ensure the wells are washed according to development wells the guidance of washing and residue washing buffer is removed as much as possible Delayed color development or Ensure color development solution or stop delayed stopping of color solution is added sequentially and is development in the wells 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 Histone Extract Preparation OP 0006 EpiQuik Total Histone Extraction Kit Modified Histone H4 Assy P 3062 EpiQuik Total Histone H3 Quantification Kit Colorimetric P 3073 EpiQuik Total Histone H4 Quantification Kit Fluorometric P 4023 EpiQuik Global Acetyl Histone H4 K5Quantification Kit Fluorometric P 4024 EpiQuik Global Acetyl Histone H4 K8Quantification Kit Colorimetric P 4025 EpiQuik Global Acetyl Histone H4 K8Quantification Kit Fluorometric P 4026 EpiQuik Global Acetyl Histone H4 K16 Quantification Kit Colorimetric P 4027 EpiQuik Global Acetyl Histone H4 K16 Quantification Kit Fluorometric P 4028 EpiQuik Global Acetyl Histone H4 K12 Quantification Kit Colorimetric P 4029 EpiQuik Global Acetyl Histone H4 K12 Quantification Kit Fluorometric 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 10 Tel 1 877 374 4368 m Fax 1 7
6. 18 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072
7. OD H4 ng mg protein _ x 1000 ng mg p Slope x Protein Amount ug Histone extract added into sample wells at step 2d 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 C1 2 5 ml 20 ml 40 ml 120 ml 240 ml C2 50 ul 400 ul 800 ul 2400 ul 4800 ul Standard control N A N A 4 uL optional 8ul 8 ul Diluted C3 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted Signal 50 ul 400 ul 800 ul 2400 ul 4800 ul Reporter Diluted Enhancer 50 ul 400 ul 800 ul 2400 ul 4800 ul C4 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml c5 0 05mi 0 4 ml 0 8 ml 2 5 ml 5ml SUGGESTED STRIP WELL SETUP Table 2 The suggested strip well plate setup for H4 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 SC 1ng SC 1ng Sample Sample Sample Sample 03 SC 2ng SC 2ng Sample Sample Sample Sample D SC 5ng SC 5ng Sample Sample Sample Sample E SC 10 ng SC 10 ng Sample Sample Sample Sample F SC 20 ng SC 20 ng Sample Sample Sample Sample G
8. k provides a series of kits used for quantifying all sites degrees of histone H4 modification For added convenience and more quantitative interpretation of results we provide here the EpiQuik Total Histone H4 Quantification Kit Colorimetric This kit is designed for quantifying levels of histone H4 proteins independent of its modified state and can also be used for normalizing the modified histone H4 content of samples when run in parallel with Epigentek histone modification quantification kit series The kit has the following features 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 histone H4 with the detection limit as low as 0 5 ng well and a detection range from 50 ng to 1 ug well of histone extracts e The control is conveniently included for the quantification of total histone H4 e Strip microplate format makes the assay flexible manual or high throughput 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 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072 e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik Total Histone H4 Quantification Kit Colorimetric is designed for
9. length 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 Total Histone Calculation Calculate the average duplicate readings for the sample wells and blank wells Calculate histone H4 change using the following formula jim Treated Tested Sample OD Blank OD x 100 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 O 5 DL H4 X 1008 508 0 9 0 1 For accurate calculation 1 Generate a standard curve and plot OD value versus amount of Standard Control at each concentration point 2 Determine the slope as OD ng You can use Microsoft Excel statistical functions for slope calculation Use the most linear part of the standard curve inculding at least 4 points then calculate the amount of histone H4 using the following formulas 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 Epigentek Group Inc All rights reserved Products are for research use only Printed 2014 09 22 P 3072 Sample OD Blank
10. measuring total histone H4 amount In an assay with this kit the histone proteins are stably spotted on the strip wells The histone H4 can be recognized with a high affinity antibody and detected with a signal reporter followed by a color development reagent The ratio of histone H4 is proportional to the intensity of absorbance The absolute amount of histone H4 can be quantitated by comparing to the standard control F 0 9819 a Tissue disaggregation ij or cell lysis E c S 4 Q r i 0 ij Histone extracts 6 Histone H4 bound to assay wells o 19 fe es Histone H4 protein ng Illustrated standard curve generated with H4 standard Add detection antibody f after wash p Add color developing 1 solution for color development and 5 measure absorbance n 5 0 6 Q e 0 4 Schematic procedure of the EpiQuik Total Histone H4 Quantification Kit Colorimetric SA o O 20 40 60 80 100 120 140 160 180 200 Histone extract ng Histone nuclear extracts were prepared from MDA 231 cells using EpiQuik Total Histone Extraction Kit Cat OP 0006 and the ODs generated from histone H4 are measured 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 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072 PROTOCOL For the best results please read the protoc
11. mingdale 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 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3072 48 Cat P 3072 96 Upon Receipt C1 10X Wash Buffer 14 ml 28 ml 47 C2 Histone Assay Buffer 4ml 8 ml 4 C3 Capture Antibody 1000X 5 ul 10 pl 4 C4 Color Developer 5 ml 10 ml 4 C5 Stop Solution 5 ml 10 ml RT Standard control 50 ug ml 10 pl 20 ul 20 C Signal Reporter 2000X 6 ul 12 ul 20 C Enhancer Solution 6 ul 12 ul 20 C 8 Well Assay Strips With Frame 6 12 47 User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store Standard Control Signal Reporter and Enhancer Solution at 20 C away from light 2 Store C1 C2 C3 C4 and 8 Well Assay Strips at 4 C away from light and 3 Store remaining components at room 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 C1 10X Wash Buffer contains salt precipitates before use If so warm at room temperature or 37 C and shake the buffer u
12. move 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 Blank Wells Add 50 ul of C2 to each blank well c Standard Wells Add 49 ul of C2 and 1 ul of Diluted Standard Control to each standard well with a minimum of six wells each at a different concentration between 1 and 50 ng ul based on the dilution chart in Step 1e see Table 2 under the Suggested Strip Well Setup section as an example d Sample Wells Add 46 to 49 ul of C2 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 of histone extract per well e Tightly cover strip well microplate with Parafilm M to avoid evaporation and incubate at 37 C for 90 to 120 min f Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted C1 1X Wash Buffer each time 3 Antibody Binding and Signal Enhancing a Add 50 ul of the Diluted C3 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted C3 solution from each well c Wash each well three times with 150 ul of the Diluted C1 1X Wash Buffer each time d Add 50 ul of the Diluted Signal Reporter to each well then cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min e Remove the Diluted Signal Reporter solu
13. ns 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 Total Histone H4 Quantification Kit Colorimetric is for research use only and is not intended for diagnostic or therapeutic application A BRIEF OVERVIEW Histone H4 along with H2A H2B and H3 is involved in the structure of chromatin in eukaryotic cells Histone H4 can undergo several different types of epigenetic modifications that influence cellular processes such as transcription activation inactivation chromosome packaging and DNA damage repair These modifications including acetylation and methylation occur on the N terminal tail domains of histone H4 through catalyzation of histone modifying enzymes This results in the remodeling of the nucleosome structure into an open conformation which is more accessible to transcription complexes Thus quantitative detection of various histone modifications would provide useful information for better understanding epigenetic regulation of cellular processes and for developing HMT targeted drugs Epigente
14. ntil the salts are re dissolved and 2 check if a blue color is present in C4 Color Developer which would indicate a contamination of the solution and should not be used To avoid contamination transfer the amount of C4 required into a secondary container tube or vial before adding C4 into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED O Adjustable pipette or multiple channel pipette Aerosol resistant pipette tips 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Distilled water Oo 0 0 0 0 0 O Histone extracts 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 Multiple channel pipette reservoirs Microplate reader capable of reading absorbance at 450 nm Page 2 Printed 2014 09 22 P 3072 O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik Total Histone H4 Quantification 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 suggestio
15. ol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of histone extracts for each assay can be between 50 ng and 1 ug with an optimal range of 0 1 to 0 2 ug 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 C1 1X Wash Buffer 48 Assay Kit Add 13 ml of C110X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of C1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted C1 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare Diluted C3 Capture Antibody Solution Dilute C3 Capture Antibody with Diluted C1 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of C3 to 1000 ul of Diluted C1 50 ul of Diluted C3 will be required for each assay well c Prepare Diluted Signal Reporter Solution Dilute Signal Reporter with Diluted C1 1X Wash Buffer at a ratio of 1 2000 i e add 1 ul of Signal Reporter to 2000 ul of Diluted C1 50 ul of Diluted Signal Reporter will be required for each assay well d Prepare Diluted Enhancer Solution Dilute Enhancer Solution with Diluted C11X Wash Buffer at a ratio of 1 5000 i e add
16. tion from each well 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 m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3072 Wash each well four times with 150 ul of the Diluted C1 1X Wash Buffer each time Add 50 ul of the Diluted Enhancer to each well then carefully cover with Parafilm M or aluminum foil and incubate at room temperature for 30 min Remove the Diluted Enhancer from each well Wash each well with 150 ul of the Diluted C1 each time for five times 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 C4 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 C4 solution will turn blue in the presence of sufficient histone H4 Add 50 ul of C5 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 C5 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 automatically subtract reference wavelength absorbance from the test wave

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