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1. EpiQuik Total Histone H4 Quantification Kit Fluorometric Base Catalog P 3073 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik Total Histone H4 Quantification Kit Fluorometric 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 500 ng 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 F
2. Blank Wells Add 50 ul of F2 to each blank well c Standard Wells Add 49 ul of F2 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 F2 and 1 ul 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 min to 120 min f Remove the reaction solution from each well Wash each well three times with 150 ul of the Diluted F1 1X Wash Buffer each time 3 Antibody Binding and Signal Enhancing a Add 50 ul of the Diluted F3 to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min b Remove the Diluted F3 solution from each well c Wash each well three times with 150 ul of the Diluted F1 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 solution from each well 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 48
3. 400 ul 800 ul 2400 ul 4800 ul Reporter Diluted Enhancer 50 ul 400 ul 800 ul 2400 ul 4800 ul Fluorescence 0 05 ml 0 4ml 0 8 ml 2 4 ml 4 8 ml Development Solution 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 c 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 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 Printed 2014 09 22 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
4. and detected with a signal reporter followed by a fluorescence development reagent The ratio of histone H4 is proportional to the intensity of fluorescence The absolute amount of histone H4 can be quantified by comparing to the standard control E Tissue disaggregation 16000 or cell lysis G gt p e s 5 ij Histone extracts o ro 0 K Histone H4 bound o to assay wells L 6 2 Add detection antibody T after wash oa G Add fluoro developing solution for fluorescence 0 T T T 1 development and 0 5 10 15 20 measurement Histone H4 protein ng Schematic procedure of the EpiQuik Total Illustrated standard curve generated with H4 standard Histone H4 Quantification Kit Fluorometric 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 Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3073 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 50 ng and 1 ug with an optimal range of 0 1 to 0 2 ug Histone Extraction You can use your own method of choice when preparing histone extracts from treated and untreated samples Epigentek also offers a histone extraction kit Cat OP 0006 opti
5. 4 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 3073 Wash each well four times with 150 ul of the Diluted F1 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 F1 1X Wash Buffer 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 50 ul of Fluorescence Development Solution to each well and incubate at room temperature Continue to monitor the development approximately 2 4 minutes The color in the standard wells containing the higher concentrations may turn slightly pink during this period Read the fluorescence on a fluorescence microplate reader within 2 to 10 min at 530ex 590em nm Note If the stripwell microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 5 Total Histone 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 f
6. Control Suggested Standard Curve Preparation First dilute Standard Control with F2 Histone Assay Buffer to 50 ng ul by adding 5 ul of Standard Control to 5 ul of F2 Histone Assay Buffer Then further prepare five concentrations by combining the 50 ng ul Diluted Standard Control with F2 Histone Assay 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 m Web www epigentek com Printed 2014 09 22 Epigentek Group Inc All rights reserved Products are for research use only P 3073 Resulting SC Tube G0 Sh pe Concentration 1 1 0 ul 49 0 ul 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 ul Note Keep each of the diluted solutions except Diluted F1 1X Wash Buffer on ice until use Any remaining diluted solutions other than Diluted F1 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 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
7. armingdale 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 3073 KIT CONTENTS Component 48 Assays 96 Assays Storage Cat P 3073 48 Cat P 3073 96 Upon Receipt F1 10X Wash Buffer 14 ml 28 ml 4 F2 Histone Assay Buffer 4ml 8 ml 47 F3 Detection Antibody 1000X 5 ul 10 pl 4 F4 Fluoro Developer 12 ul 24 ul 20 C F5 Fluoro Enhancer 12 ul 24 ul 20 C F6 Fluoro Diluter 4ml 8 ml 4 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 4 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 parts on frozen ice packs at 4 C Upon receipt 1 Store F4 F5 Standard Control Signal Reporter and Enhancer Solution at 20 C away from light 2 Store F1 F2 F3 F6 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 F1 10X Wash Buffer contains salt precipitates before use I
8. duct 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 Fluorometric 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 the 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 Epigentek 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 E
9. f so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED oO 0 0 O 0 0 0 O Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Fluorescence microplate reader capable of reading fluorescence at 530ex 590em nm 1 5 ml microcentrifuge tubes Incubator for 37 C incubation Distilled water 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 Page 2 Printed 2014 09 22 P 3073 O Parafilm M or aluminum foil GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik Total Histone H4 Quantification Kit Fluorometric 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 Pro
10. 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 fluorescence reading Check if appropriate fluorescence wavelength 530ex 590em 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 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 fluorescence Decrease the development time in Step 4a No signal or weak signal only in sample wells Protein sample is not prope
11. mized 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 F1 1X Wash Buffer 48 Assay Kit Add 13 ml of F1 10X Wash Buffer to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of F1 10X Wash Buffer to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted F1 1X Wash Buffer can now be stored at 4 C for up to six months b Prepare Diluted F3 Detection Antibody Solution Dilute F3 Detection Antibody with Diluted F1 1X Wash Buffer at a ratio of 1 1000 i e add 1 ul of F3 to 1000 ul of Diluted F1 50 ul of Diluted F3 will be required for each assay well c Prepare Diluted Signal Reporter Solution Dilute Signal Reporter with Diluted F1 1X Wash Buffer at a ratio of 1 2000 i e add 1 ul of Signal Reporter to 2000 ul of Diluted F1 50 ul of Diluted Signal Reporter will be required for each assay well d Prepare Diluted Enhancer Solution Dilute Enhancer Solution with Diluted F1 1X Wash Buffer at a ratio of 1 5000 i e add 1 ul of Enhancer Solution to 5000 ul of F1 About 50 ul of this Diluted Enhancer will be required for each assay well e Prepare Fluorescence Development Solution Add 1 ul of F4 Fluoro Developer and 1 ul of F5 Fluoro Enhancer to every 500 ul of F6 Fluoro Diluter About 50 ul of Fluorescence Development Solution will be required for each well to be developed f Prepare Diluted Standard
12. ntification Kit Colorimetric P 3072 EpiQuik Total Histone H4 Quantification Kit Colorimetric 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 1 718 484 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 3073
13. or the sample wells and blank wells Calculate histone H4 change using the following formula H Treated Tested Sample RFU Blank RFU x 100 Untreated Control Sample RFU Blank RFU Example calculation Average RFU of treated sample is 6000 Average RFU of untreated control is 11000 Average RFU of blank is 1000 6000 1000 H4 xX 100 50 11000 1000 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 Sample RFU Blank RFU H4 ng mg protein x 1000 ng mg p Slope x Protein Amount ug Histone extract added into sample wells at step 2d Page 7 Printed 2014 09 22 P 3073 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 F1 2 5 ml 20 ml 40 ml 120 ml 240 ml F2 50 ul 400 ul 800 ul 2400 ul 4800 ul Standard Control N A N A 4 uL optional 8ul 8 ul Diluted F3 50 ul 400 ul 800 ul 2400 ul 4800 ul Diluted Signal 50 ul
14. piQuik Total Histone H4 Quantification Kit Fluorometric 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 fluorometric assay without the need for radioactivity electrophoresis or chromatography e Specifically captures histone H4 with the detection limit as low as 0 2 ng well and a detection range from 50 ng to 500 ng 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 3073 e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik Total Histone H4 Quantification Kit Fluorometric is designed for 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
15. rly 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 for research use only Page 9 Printed 2014 09 22 P 3073 Uneven Insufficient washing of the Ensure the wells are washed according to fluorescence wells the guidance of washing and residue development washing buffer is removed as much as possible Delayed fluorescence Ensure fluorescence development is added development n the wells 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 Histone Extract Preparation OP 0006 EpiQuik Total Histone Extraction Kit Modified Histone H4 Assy P 3062 EpiQuik Total Histone H3 Qua

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