EpiQuik™ Global Histone H4 Acetylation Assay Kit
Contents
1. EpiQuik Global Histone H4 Acetylation Assay Kit Base Catalog P 4009 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Global Histone H4 Acetylation Assay Kit is suitable for specifically measuring global histone H4 acetylation using a variety of mammalian cells including fresh and frozen tissues and cultured adherent and suspension cells 110 Bi County Blvd Ste 122 Farmingdale NY 11735 P 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 201 ARo Epigentek Group Inc All rights reserved Products are for research use only P 4009 KIT CONTENTS Components 48 assays 96 assays P 4009 48 P 4009 96 GE1 10X Lysis Buffer 5 ml 10 ml GE2 Extraction Buffer 8 ml 16 ml GE3 10X Wash Buffer 14 ml 28 ml GE4 Histone Buffer 0 5 ml l ml GE5 Blocking Buffer 10 ml 20 ml GE6 Antibody Buffer 6 ml 12 ml GE7 Capture Antibody 100 ug ml 25 ul 50 ul GE8 Detection Antibody 400 ug ml 10 ul 20 ul GE9 Developing Solution 5 ml 10 ml GE10 Stop Solution 3 ml 6 ml Acetylated Histone H4 Control 60 ug ml 10 ul 20 ul 8 Well Assay Strips with Frame 6 12 User Guide Formaximum recovery of the products centrifuge the original vial priorto opening the cap SHIPPING amp STORAGE The kit is shipped in two parts one part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store GE8 and Acetylated His2. Dilute GE1 with distilled water pH 7 2 to 7 5 at a 1 10 ratio e g 1 ml of GE1 9 ml of distilled water Add 1 ml of the diluted GE1 per every 200 mg of tissue and disaggregate tissue pieces by 10 30 strokes 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 Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 minutes at 4 C If total mixture volume is less than 2 ml transfer mixture to a 2 ml vial and centrifuge at 10 000 rpm for 1 minute at 4 C Remove supernatant For Adherent Cells Cells treated or untreated are grown to 70 80 confluency then trypsinized and collected into a 15 ml conical tube Count cells in a hemacytometer Centrifuge the cells at 1000 rpm for 5 minutes and discard the supernatant Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 minutes Discard the supernatant Add diluted GE1 to re suspend cell pellet 200 ul 1 x 10 cells Transfer cell suspension to a 1 5 ml vial and incubate on ice for 5 minutes and vortex occasionally Pellet cell debris by centrifuging at 12 000 rpm for 30 seconds For Suspension Cells Collect cells treated or untreated into a 15 ml conical tube 1 2 x 10
3. cells are required for each reaction Count cells in a hemacytometer Centrifuge the cells at 1000 rpm for 5 minutes and discard the supernatant Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 minutes Discard the supernatant Add diluted GE1 to re suspend cell pellet 100 pl 1 x 10 cells Transfer cell suspension to a 1 5 ml vial and incubate on ice for5 minutes and vortex occasionally Pellet cell debris by centrifuging at 12 000 rpm for 30 seconds Histone Extraction Add glycerol to GE2 ata 1 10 ratio e g add 100 ul of glycerol to 900 ul of GE2 to prepare the GE2 Glycerol Solution Add the diluted GE1 to cell debris 10 ul 1 x 10 cells or 40 mg of tissue followed by adding 3 volumes of GE2 Glycerol Solution Mix by vortexing and incubate on ice for 5 minutes Pellet nucleic debris by centrifuging at 12 000 rpm for 5 minutes at 4 C Transfer the supernatant toa 1 5 ml vial Add 100 TCA to the supernatant ata 1 4 ratio ex add 100 ul of TCA to 300 ul of supernatant Final concentration of TCA should be 25 Incubate on ice for 30 minutes Collect the precipitate by centrifuging at 12 000 rpm for 10 minutes at 4 C Remove supernatant and add 1 ml of acetone containing 0 1 HCI to precipitate Mix and incubate on ice for 1 minute Collect the pellet by centrifuging at 12 000 rpm for 2 minutes at 4 C Wash the pellet with 1 ml of acetone Allow 1 minute on ice for wash Collect the pellet by centrifugin
4. extract contains a sufficient amount of protein Ensure the protein extracts are stored at 80 C for no more than 3 months Check if wash at each step is performed according to the protocol Ensure the well is not contaminated from adding 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 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 control Overdevelopment RELATED PRODUCTS P 3015 EpiQuik P 3016 EpiQuik P 3017 EpiQuik P 3018 EpiQuik P 4008 EpiQuik 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 the control protein or by using control protein contaminated tips Decrease development time in Step 9 of Histone H4 Acetylation Detection In Situ Histone H3 K4 Methylation Assay Kit In Situ Histone H3 K9 Methylation Assay Kit Global Histone H3 K4 Methylation Assay Kit Global Histone H3 K9 Methylation Assay Kit Global Histone H3 Acetylation Assay Kit Page 8 Printed 2014 10 06 P 4009
5. e blank Amount ng mg protein _ _ x 1000 slope Page 6 TROUBLESHOOTING No Signal for Both the Positive Control and the Samples Reagents are added incorrectly The well is not completely dried The well is incorrectly washed before protein coating Incubation time and temperature is incorrect Check if reagents are added in order and if any steps of the procedure may have been omitted by mistake Ensure the well is incubated without humidity and dried before adding GES Blocking Buffer Ensure the well is not washed before adding the positive control or protein extracts Ensure the incubation time and temperature described in the protocol are followed correctly No Signal or Very Weak Signal for Only the Positive Control The positive control is added insufficiently The positive control degraded due to incorrect storage No Signal for Only the Sample The protein sample is not properly extracted The protein amount is added into well insufficiently Protein extracts are incorrectly stored or have been stored for a long period High Background Present for the Blank The well is not washed sufficiently Contaminated by the positive Ensure a sufficient amount of control is added to the well Follow the guidance in the protocol for storage of the positive control Follow the protocol instructions for the histone protein extraction Ensure
6. g at 12 000 rpm for 2 minutes at 4 C Remove the supernatant as much as possible and air dry the pellet for 5 minutes Add distilled water to dissolve pellet 10 ul of water per amount of pellet extracted from 1 x 10 cells or 40 mg of tissue and measure histone protein concentration The histone extract can be used immediately or stored at 80 C 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 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 a NEO 10 11 110 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 Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 Histone H4 Acetylation Detection Determine the number of strip wells required Leave these strips in the plate frame remaining unused strips can be placed back in the bag Seal the bag tightly and store at 4 C Dilute GE3 with distilled water oH 7 2 7 5 ata 1 10 ratio Adjust protein concentration to 200 ng ul or 400 ng ul with GE4 and add 5 ul 1 2 ug of the protein solution into the central area of each well Spread out the solution over the strip well surtace by pipetting the solution up and down several times Incubate the strip wells at 37 C with no humidity for 60 90 minutes to evaporate the solut
7. ion and dry the wells For the blank add 5 ul of GE4 to the wells For the positive control dilute Acetylated Histone H4 Control to 2 30 ng ul with GE4 and add 5ul 10 150 ng of the diluted Acetylated Histone H4 Control solution to the wells Add 150 pl of GES to the dried wells and incubate at 37 C for 30 minutes Aspirate and wash the wells with 150 ul of diluted GE3 three times Dilute GE7 ata 1 100 ratio to 1 ug ml with GE6 Add 50 ul of diluted GE7 to the wells and incubate at room temperature for 60 minutes on an orbital shaker 50 100 rpm Aspirate and wash the wells with 150 ul of diluted GE3 four times Dilute GE8 ata 1 1000 ratio to 0 4 ug ml with GE6 Add 50 ul of diluted GE8 to the wells and incubate at room temperature for 30 minutes Aspirate and wash the wells with 150 ul of diluted GE3 four times Allow 3 minutes for the last wash Add 100 ul of GE9 to the wells and incubate at room temperature for 2 10 minutes away from light Monitor the color development in the sample and standard wells blue Add 50 ul of GE10 to the wells and read absorbance on microplate reader at 450 nm Calculate Histone H4 acetylation OD sample blank Acetylation _ _ x 100 OD untreated control blank For accurate calculation plot OD value versus amount of Acetylation Histone H4 Control and determine the slope as delta OD ng Calculate the amount of acetylation H4 using the following formula OD sampl
8. le reliable and consistent assay conditions Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 PRINCIPLE amp PROCEDURE The EpiQuik Global Histone H4 Acetylation Assay Kit is designed for measuring global histone H4 acetylation In an assay with this kit the histone proteins are stably spotted on the strip wells The acetylated histone H4 can be recognized with a high affinity antibody The ratio or amount of acetylated H4 can be quantified through HRP conjugated secondary antibody color development system and is proportional to the intensity of color development e Tissue disaggregation or cell lysis a Histone extraction G ES Histone coating onto assay wells Add capture antibody after wash amp gt a Add detection antibody after wash Soe 5 lt a Add developing solution SS for developing color and measure absorbance Fig1 Schematic Procedure for Using the EpiQuik Global Histone H4 Acetylation Assay Kit PROTOCOL Nucleic Extraction Preparation For Tissue Samples Place the tissue sample into a 60 or 100 mm plate Remove unwanted tissue such as fat and necrotic material from the sample Weigh the sample and cut the sample into small pieces 1 2 mm with a scalpel or scissors Transfer tissue pieces to a Dounce homogenizer
9. s performance and design Intellectual Property EpiQuik is a trademark of Epigentek Inc The EpiQuik Global Histone H4 Acetylation Assay Kit and method of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Acetylation of histones including histone H4 has been involved in the regulation of chromatin structure and the recruitment of transcription factors to gene promoters Histone acetyltransterases HATs and histone deacetylases HDACs play critical roles in controlling histone acetylation Histone acetylation is tightly involved in cell cycle regulation cell proliferation and apoptosis Reversible acetylation of nucleosomal histones H4 generally is believed to be correlated with potential transcriptional activity of eukaryotic chromatin domains Histone H4 acetylation may be increased by inhibition of HDACs and decreased by HAT inhibition There are very few methods used for measuring global histone H4 acetylation The EpiQuik Global Histone H4 Acetylation Assay Kit addresses this problem by providing a unique procedure to measure global acetylation of histone H4 The kit has the following features e Quick and efficient procedure which can be finished within 5 hours e Innovative colorimetric assay without the need for radioactivity electrophoresis or chromatography e Strip microplate format makes the assay flexible manual or high throughput e Simp
10. tone H4 Control at 20 C away from light 2 Store GE3 GE5 GE6 GE7 GE9 and 8 Well Assay Strips at 4 C away from light 3 Store all other components at room temperature The components of the kit are stable for up to 6 months when stored properly Note Check if wash buffer GE3 contains salt precipitates before using If so warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Orbital Shaker Pipettes and Pipette Tips Microplate Reader 1 5 ml Microcentrifuge Tubes 60 or 100 mm Plate 0000 oa Dounce Homogener O 100 TCA Solution 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 2 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 4009 O Glycerol O Acetone O 5 HCl O Distilled Water GENERAL PRODUCT INFORMATION Usage Limitation The EpiQuik Global Histone H4 Acetylation Assay Kit is for research use only and is not intended for diagnostic or therapeutic application Safety Suitable lab coat disposable gloves and eye protection is required when working with the kit Quality Control Epigentek guarantees the performance of all products in the manner described in our product instructions Product Updates Epigentek reserves the right to change or modify any product to enhance it
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