EpiQuik™ Tissue Chromatin Immunoprecipitation Kit
Contents
1. Forward 20 uM 10 ul 15 ul 20 ul Reverse 20 uM 10 ul 15 ul 20 ul 8 Well Assay Strips with Frame 3 6 12 8 Well Strip Caps 3 6 12 F Spin Column 30 50 100 F Collection Tube 30 50 100 User Guide 1 Spin the solution down to the bottom prior to use Extra volume is included for optimization purpose of sonication conditions if necessary SHIPPING amp STORAGE The kit is shipped in three pars the first and second parts at ambient room temperature and the third part on frozen ice packs at 4 C Upon receipt 1 Store the following components at 4 C Protease Inhibitor Cocktail Normal Mouse IgG Anti RNA Polymerase II Proteinase K Control Primers and 8 Well Assay Strips 2 Store all other components at room temperature The kit is stable for up to 6 months from the shipment date when stored properly 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 Variable temperature waterbath O Vortex mixer O Desktop centrifuge up to 14 000 rpm O Sonicator Page 2 Printed 2014 10 06 P 2003 Dounce homogener Orbital shaker Pipettes and pipette tips 1 5 ml microcentrifuge tubes 15 ml conical tube Antibody of interest for chromatin immunoprecipitation 37 formaldehyde Glycine solution TE2. EpiQuik Tissue Chromatin Immunoprecipitation Kit Base Catalog P 2003 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik Tissue Chromatin Immunoprecipitation Kit is suitable for combining the specificity of immunoprecipitation with qualitative and quantitative PCR MS PCR DNA sequencing and southern blot as well as DNA microarray Like using other ChIP kits if you use the EpiQuik ChIP kits choice of good antibody i e IP proven is required for precipitating the fixed protein DNA complexes 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 m Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 2003 KIT CONTENTS Components 24 reactions 48reactions 96 reactions P 2003 1 P 2003 2 P 2003 3 CP 1 Wash Buffer 28 ml 2 x 28 ml 4 x 28 ml CP2 Antibody Buffer 15 ml 30 ml 2 x 30 ml CP3 Lysis Buffer 4 ml 6 ml 10 ml CP4 ChIP Dilution Buffer 4 ml 6 ml 10 ml CP5 DNA Release Buffer 1 ml 2x2mi 2x2 ml CP6 Reverse Buffer 1 ml 2x2ml 2x2ml CP7 Binding Buffer 5ml 8 ml 15 ml CP8 Elution Buffer 0 6 ml 1 2 ml 2 ml Homogenizing Buffer 5 ml 10 ml 16 ml Protease Inhibitor Cocktail 100X 25 ul 50 ul 100 ul Normal Mouse IgG 1 mg ml 104l 15 ul 25 pl Anti RNA Polymerase II Img ml 5 ul 7 ul 11 ul Proteinase K 10 mg ml 25 ul 50 ul 100 ul Contro Primers GAPDH
3. info epigentek com Web www epigentek com Printed 2014 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 2003 110 Bi County Blvd Ste 122 Farmingdale NY 11735 equipment If desired remove 5 ul of sonicated cell lysate for agarose gel analysis The length of sheared DNA should be between 200 1000 bp Pellet cell debris by centrifuging at 14 000 rpm for 10 minutes at 4 C Protein DNA Immunoprecipitation Transfer supernatant to a 1 5 ml vial supernatant can be stored at 80 C at this step Dilute the required volume of supernatant with CP4 ata 1 1 ratio ex add 100 ul of CP4 to 100 ul of cell supernatant Remove 5 ul of the diluted supernatant to a 0 5 ml vial Label the vial as input DNA and place on ice Remove the incubated antibody solution and wash the strip wells three times with 150 ul of CP2 by pipetting in and out Transfer 100 ul of the diluted supernatant to each strip well Cover the strip wells with Parafilm M and incubate at room temperature 22 25 C for 60 90 minutes on an orbital shaker 50 100 rom Remove supernatant Wash the wells with 150 ul of CP1 six times Allow 2 minutes on an orbital shaker 100 rpm for each wash Wash the wells once for 2 minutes with 150 ul of 1X TE Buffer Cross Linked DNA Reversal DNA Purification Add 1 ul of Proteinase K to each 40 ul of CP5 and mix Add 40 ul of CP5 containing Proteinase K to the samples including the
4. input DNA vial Cover the sample wells with strip caps and incubate at 65 C in a waterbath for 15 minutes Add 40 ul of CP6 to the samples mix re cover the wells with strip caps and incubate at 65 C in a waterbath for 90 minutes Also add 40 ul of CP6 to the vial containing supernatant labeled as input DNA Mix and incubate at 65 C for 90 minutes Place a spin column into a 2 ml collection tube Add 150 ul of CP7 to the samples and transfer mixed solution to the column Centrifuge at 12 000 rpm for 20 seconds Add 200 ul of 70 ethanol to the column centrifuge at 12 000 rpm for 15 seconds Remove the column from the collection tube and discard the flowthrough Replace the column to the collection tube Add 200 ul of 90 ethanol to the column and centrifuge at 12 000 rpm for 20 seconds Remove the column and discard the flowthrough Replace column to the collection tube and wash the column again with 200 ul of 90 ethanol at 12 000 rpm for 35 seconds Place the column in a new 1 5 ml vial Add 10 20 ul of CP8 directly to the filter in the column and centrifuge at 12 000 rpm for 20 seconds to elute purified DNA DNA is now ready for use or storage at 20 C Note Ifa conventional PCR or a SYBR green real time PCR is performed contro primers 110 bp for human tissues included in the kit can be used as the positive control For mouse or rat tissues Control primers may be needed to be designed by the user For conventional PCR
5. the number of PCR cycles may need to be optimized for better PCR results 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 10 06 Epigentek Group Inc All rights reserved Products are for research use only P 2003 TROUBLESHOOTING Little or No PCR Products Insufficient amount of tissue 2 Insufficient or too much cross linking 3 Insufficient tissue lysis 4 Insufficient too much sonication 5 Antibody does not bind to protein 6 Incorrect temperature insufficient time for DNA release and reversal of cross linking 7 Incorrect PCR conditions 8 Wrong or bad primers 9 The column is not washed with 90 ethanol 10 DNA is not completely passed through the filter Increase tissue amount ex gt 10 mg of tissue per reaction Check if the appropriate cross link step is carried out according to the protocol Follow the guidelines in the protocol Check the tissue lysis by observing a 5 ul portion of the tissue lysate under the microscope Follow the protocol instructions for obtaining the appropriate sized DNA Keep the sample on ice during the sonication Check if the subclass or isotype of the antibody is correct Choose an antibody that is ChIP or IP grade Follow the guidelines in the protocol for appropriate temperature and time Check if all PCR components are added Increase amount of DNA added to PCR rea
6. Tissue Methyl Histone H3 K9 ChIP Kit Tissue Methyl Histone H3 K4 ChIP Kit Acetyl Histone H3 ChIP Kit Acetyl Histone H4 ChIP Kit Methyl Histone H3 K27 ChIP Kit Tissue Methyl Histone H3 K27 ChIP Kit Methyl CpG Binding Domain Protein 2 ChIP Kit Tissue Methyl CpG Binding Domain Protein 2 ChIP Kit Page 8 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 2003
7. buffer pH 8 0 Ethanol 96 100 NnOOdaangzgaaoaaoo GENERAL PRODUCT INFORMATION 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 its performance and design Usage Limitation The EpiQuik ChIP kits are for research use only and are not intended for diagnostic or therapeutic application Intellectual Property The EpiQuik ChIP kits and method of contain proprietary technologies by Epigentek EpiQuik is a trademark of Epigentek Group Inc A BRIEF OVERVIEW 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 Protein DNA interaction play a critical role for cellular functions such as signal transduction gene transcription chromosome segregation DNA replication and recombination and epigenetic silencing Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein DNA interaction is important for understanding cellular process Chromatin Immunoprecipitation ChIP offers an advantageous tool for studying protein DNA interactions Unlike other methods such as EMASA DNA microarrays and report gene assays which analyze direct interactions between protein and
8. DNA in vitro ChIP can detect that a specific protein binds to the specific sequences of a gene in living cells A typical ChIP includes the following steps 1 formaldehyde cross link of chromatin 2 shearing of chromatin 3 immunoprecipitation 4 reversal of cross link and 5 DNA purification Requirement for using ChIP to analyze protein DNA interaction in solid tissues and tumors is rapidly increasing There are several methods used for chromatin immunoprecipitation however most of these methods available so far are very time consuming labor intensive low throughput and furthermore not specifically designed for solid tissues and tumors Page 3 Printed 2014 10 06 P 2003 The EpiQuik ChIP kits use a proprietary and unique procedure composition to investigate protein DNA interaction in solid tissues and tumors The EpiQuik ChIP kit series have the following features e The fastest procedure available which can be finished within 5 hours e Strip microplate format makes the assay flexible manual or high throughput e Columns for DNA purification are included save time and reduce labor e Compatible with all DNA amplification based approaches e Simple reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik Tissue Chromatin Immunoprecipitation Kit contains all reagents required for carrying out a successful chromatin immunoprecipitation from mammalian tissues Particularly this kit in
9. Note Always cap spin columns before placing them in the microcentrifuge 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Before starting perform the following Prepare the following required solutions not included 90 Ethanol 70 Ethanol 1 25 M Glycine Solution Ensure that all buffers are in clear solution Shake or vortex if these buffers precipitate Antibody Binding to the Assay Strip Wells 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 Wash strip wells once with 120 ul of CP1 Add 100 ul of CP2 to each well and then add the antibodies 1 ul of Normal Mouse IgG as the negative control 1 ul of Anti RNA Polymerase II as the positive control and 2 3 ug of your antibody of interest Cover the strip wells with Parafilm M and incubate at room temperature for 60 90 minutes Meanwhile prepare the cell extracts as described in the next steps Tissue Disaggregation and In Vivo Cross Link 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 15 ml conical tube Prepare the Cross Link Solution by adding formaldehyde to culture medium final concentration is 1 Ex add 270 ul of 37 formaldehyde to 10 ml of cultu
10. cludes a positive control antibody RNA polymerase ll a negative control normal mouse IgG and GAPDH primers that can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol RNA polymerase Il is considered to be enriched in the GAPDH gene promoter that is expected to be undergoing transcription in most growing mammalian cells and can be immunoprecipitated by RNA polymerase II but not by normal mouse IgG In this ChIP cells are cross linked with formaldehyde and chromatin is extracted The chromatin is then sheared and added into the microwell immobilized with affinity antibodies Cross linked DNA is released from antibody captured protein DNA complex reversed and purified through the specifically designed Fast Spin Column Eluted DNA can be used for various down stream applications Tissue disaggregation and cross link Cell lysis and DNA shearing Schematic Procedure for Using the EpiQuik Protein antibody Tissue Chromatin immunoprecipitation Immunoprecipitation Kit 4 Clean protein DNA complex H and reverse cross link Capture and cleaning Elution of DNA E PCR sequencing microarray 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 reserved Products are for research use only Printed 2014 10 06 P 2003 PROTOCOL
11. ction Increase the number of cycles for PCR reaction Ensure the designed primers are specific to the target sequence Ensure that wash solution is 90 ethanol Increase centrifuge time to 1 minute at steps 3 to 7 of Cross Linked DNA Reversal DNA Purification Little or No Amplification Difference Between the Positive Control andthe Negative Control 1 Insufficient wash at each wash step 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 Follow the protocol for appropriate wash Page 7 Printed 2014 10 06 P 2003 2 Positive control antibody is added Ensure antibody is added into the correct well into the well for the negative control by mistake 3 Too many PCR cycles RELATED PRODUCTS P 2002 EpiQuik P 2006 EpiQuik P 2007 EpiQuik P 2008 EpiQuik P 2009 EpiQuik P 2010 EpiQuik P 2011 EpiQuik P 2015 EpiQuik P 2016 EpiQuik P 2017 EpiQuik P 2018 EpiQuik 110 Bi County Blvd Ste 122 Farmingdale NY 11735 If using conventional PCR decrease the cycles to appropriate cycle number Differences between quantities of starting DNA can be measured generally within the linear PCR amplification phase Chromatin Immunoprecipitation ChIP Kit Methyl Histone H3 K9 ChIP Kit Methyl Histone H3 K4 ChIP Kit
12. re medium Add 1 ml of Cross Link Solution per every 40 mg of tissue and incubate at room temperature for 15 20 minutes on a rocking platform Add 1 ml of 1 25 M Glycine solution per every 9 ml of Cross Link Solution then mix and centrifuge at 800 rpm for 5 minutes Discard the supernatant Wash cells with 10 ml of ice cold PBS once by centrifugation at 800 rpm for 5 minutes Discard the supernatant Transfer tissue pieces to a Dounce homogenizer Add 1 ml of the Homogenizing Buffer per every 200 mg of tissue and disaggregate tissue pieces by 10 20 strokes 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 5000 rpm for 5 minutes at 4 C Remove supematant Cell Lysis and DNA Shearing Add CP3 containing Protease Inhibitor Cocktail PIC Ex 10 ul of PIC to each 1 ml of CP3 to re suspend the disaggregated tissue pellet 100 ul 20 mg of tissue Transfer cell suspension to a 1 5 ml vial 600 ul maximum for each vial and incubate 10 minutes on ice and vortex occasionally Shear DNA by sonication Usually sonicate 4 to 5 pulses of 15 to 20 seconds each at level 2 using a Branson Microtip probe followed by 30 to 40 seconds rest on ice between each pulse The conditions of cross linked DNA shearing can be optimized based on tissues and sonicator Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail
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