ChromaFlash ™ Chromatin Extraction Kit
Contents
1. Add 9 ml fresh cell culture medium containing formaldehyde with a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm For Suspension Cells Collect cells treated or untreated into a 15 ml conical tube Count cells in a hemocytometer Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3d after Step 2c Add 9 ml fresh cell culture medium containing formaldehyde with a final concentration of 1 i e add 270 ul of 37 formaldehyde to 10 ml of cell culture medium to cells Incubate at room temperature 20 25 C for 10 min on a rocking platform 50 100 rpm For Tissues Put 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 Note For tissues that are not cross linked go directly to Step 2j after Step 2b Transfer tissue pieces to a 15 ml conical tube Prepare cross link solution by adding formaldehyde to cell culture medium with a final concentration of 1 e g add 270 ul of 37 formaldehyde to 10 ml of culture medium Add 1 ml of cross link solution for2. ChromaFlash Chromatin Extraction Kit Base Catalog P 2001 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The ChromaFlash Chromatin Extraction Kit is suitable for isolating chromatin or DNA protein complex from mammalian cells or tissues in a simple and rapid format Chromatin prepared by this kit can be used in a variety of chromatin immunoprecipitation methods It is the optimal method for chromatin required by Epigentek s one hour ChIP method using the ChromaFlash One Step ChIP Kit P 2025 or ChromaFlash One Step Magnetic ChIP Kit P 2026 The isolated chromatin can also be used in other chromatin related applications such as in vitro protein DNA binding assays and nuclear enzyme assays Starting Material and Input amount Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells fresh and frozen tissues etc The amount of cells and tissues for each preparation can be 1 x 10 to 5 x 10 cells and 10 mg to 200 mg respectively For optimal preparation the input amount should be 1 to 5 x 10 cells or 50 to 200 mg tissues A total of 100 standard extractions use 1 X10 cells or 50 mg of tissue per extraction can be performed with this kit Yield of chromatin is approximately 4 ug per 10 cells or per 50 mg tissues Precautions To avoid cross contamination carefully pipette the sample or solution into the tube vials Use aerosol barrier pipette tips
3. 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 Page 1 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Printen Peer 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 KIT CONTENTS Component 100 Preparations Storage P 2001 100 Upon Receipt 10X Lysis Buffer 11 ml RT Extraction Buffer 11 ml RT Chromatin Buffer 11 ml RT Protease Inhibitor Cocktails 1000X 110 ul 4 C User Guide 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped on frozen ice packs at 4 C Upon receipt 1 Protease Inhibitor cocktails at 4 C 2 Store remaining components at room temperature All components of the kit are stable for 6 months from the date of shipment when stored properly Note Check if any buffers contain salt precipitates before use If so shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED O Vortex mixer Dounce homogenizer Centrifuge including desktop centrifuge up to 14 000 rpm Pipettes and pipette tips 1 5 ml microcentrifuge tubes 15 ml conical tube Cell culture medium 37 formaldehyde if cross linked 1 25 M Glycine solution if
4. cross linked oO oO oO oO oO O Cells or tissues oO oO oO O 1XPBS oO Distilled water 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 2001 GENERAL PRODUCT INFORMATION Quality Control Each lot of the ChromaFlash Chromatin Extraction Kit 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 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 ChromaFlash Chromatin Extraction Kit is for research use only and is not intended for diagnostic or therap
5. the chromatin pellet 50 ul 1x10 cells 500 ul maximum for each vial Incubate the sample on ice for 10 min and vortex occasionally Resuspend the sample and sonicate 2 X 20 seconds to increase chromain extraction Allow the sample to cool on ice between sonication pulses for 30 seconds As an example sonication can be carried out with a microtip attached to Branson 450 sonifier setting at 25 power output Centrifuge at 12 000 rom at 4 C for 10 min Transfer supernatant to a new vial Add Chromatin Buffer at a 1 1 ratio e g add 100 ul of Chromatin Buffer to 100 ul of supernatant The chromatin solution can now be used immediately or stored at 80 C after aliquoting appropriately until further use Avoid multiple freeze thaw cycles Page 6 Printed 2014 09 22 110 Bi County Blvd Ste 122 Farmingdale NY 11735 P 2001 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 TROUBLESHOOTING Problem Possible Cause Suggestion Low yield of chromatin Insufficient amount of samples To obtain the best results the amount of samples should be 1 x10 to 5x10 cells or 50 to 200 mg tissues per ChIP reaction Insufficient chromatin extraction Ensure that all reagents have been added with the correct volume and in the correct order based on the sample amount Check for sample lysis under mi
6. croscope after the tissue cell lysis step Ensure that the cell or tissue species are compatible with this extraction procedure Lysis or extraction reagents have Ensure that the kit has not exceeded expired Expired reagents may the expiration date of the kit Standard cause inefficient extraction shelf life when stored properly is 6 months from date of receipt Incorrect temperature and or Ensure the incubation time and insufficient incubation time during temperature described in the protocol extraction are followed correctly Degradation of Improper storage of chromatin Chromatin sample should be stored at chromatin 80 C 3 6 months Avoid multiple freeze thaw cycles RELATED PRODUCTS Chromatin Shearing and Cleanup P 1006 DNA Concentrator Kit P 2023 ChromaFlash Chromatin Isolation and Shearing Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 ChIP Reaction P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash One Step Magnetic ChIP kit PCR Analysis P 1029 ChIP flash Quantitative PCR Fast Kit ChIP Grade Antibodies A 1001 DNMT1 Monoclonal Antibody 60B122 1 A 1003 DNMTS3A Polyclonal Antibody A 1004 DNMT3B Polyclonal Antibody Page 7 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Pgd ga a 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 Produ
7. cts are for research use only A 1006 MBD1 Polyclonal Antibody A 1007 MBD2 Polyclonal Antibody A 1008 MBD3 Polyclonal Antibody A 1009 MBD4 Polyclonal Antibody A 1010 MGMT Polyclonal Antibody A 1012 MeCP2 Polyclonal Antibody A 1014 5 Methylcytosine Monoclonal Antibody 33D3 A 2021 RING1 Polyclonal Antibody A 2029 ENX1 Polyclonal Antibody A 2030 ENX2 Polyclonal Antibody A 2032 RNA Polymerase II Monoclonal Antibody CTD4H8 A 3009 SUV39H1 Monoclonal Antibody A 3010 SUV39H2 Polyclonal Antibody A 4001 HDAC1 Monoclonal Antibody A 4002 HDAC2 Monoclonal Antibody A 4003 HDAC3 Monoclonal Antibody A 4004 HDAC4 Polyclonal Antibody A 4005 HDAC5 Polyclonal Antibody A 4006 HDACE6 Polyclonal Antibody A 4007 HDAC7 Polyclonal Antibody A 4008 HDAC8 Monoclonal Antibody A 4009 HDAC9 Polyclonal Antibody A 4012 PCAF Polyclonal Antibody A 4013 GCN5 Polyclonal Antibody A 4020 p300 Polyclonal Antibody A 4021 Acetyl Histone H3 K9 14 Polyclonal Antibody A 4022 Acetyl Histone H3K9 Polyclonal Antibody A 4023 Acetyl Histone H3K14 Polyclonal Antibody A 4024 Acetyl Histone H3K18 Polyclonal Antibody A 4025 Acetyl Histone H3K23 Polyclonal Antibody A 4026 Acetyl Histone H3K56 Polyclonal Antibody A 4027 Acetyl Histone H4K5 Polyclonal Antibody A 4028 Acetyl Histone H4K8 Polyclonal Antibody A 4031 Histone H3K4 Monomethy Polyclonal Antibody A 4032 Histone H3K4 Dimethyl Polyclonal Antibody A 4033 Histone H3K4 Trimethyl Polyclonal Antibody A 4034 H
8. eutic application A BRIEF OVERVIEW Chromatin immunoprecipitation ChIP offers an advantageous tool for studying protein DNA interaction With ChIP the experimenter can determine if a specific protein binds to the specific sequences of a gene in living cells by combining with PCR ChIP PCR microarray ChIP chip or sequencing ChIP Seq techniques For example the measurement of the amount of methylated histone H3 at lysine 9 meH3 K9 associated with a specific gene promoter region under various conditions can be achieved through a ChIP PCR assay while recruitment of meH3 K9 to the promoters on a genome wide scale can be detected by ChIP chip In particular the ChIP method with specific antibodies directly against various transcriptional factors is widely demanded For performing ChIP chromatin or DNA protein complex in cells or tissues should be first isolated The ChromaFlash Chromatin Extraction Kit addresses the inconvenience and time consuming issues of existing chromatin preparation methods by introducing the following features e Extremely fast procedure the entire procedure from cell tissue sample to ready to use chromatin is less than 60 minutes e Convenient and flexible the kit is suitable for preparing both native chromatin and cross linked chromatin from monolayer or suspension cells or from tissues e Unsheared chromatin makes it customizable for various analysis workflows that require either intact or fragmented chr
9. every 40 mg tissues Incubate at room temperature for 15 20 min on a rocking platform Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Page 5 Printed 2014 09 22 P 2001 Epigentek Group Inc All rights reserved Products are for research use only Mix and centrifuge at 800 rpm for 5 min Discard the supernatant Wash cells with 10 ml of ice cold PBS once by centrifugation at 800 rpm for 5 min Discard the supernatant Transfer tissue pieces to a Dounce homogenizer Add 1 ml Working Lysis Buffer for every 200 mg tissues Disaggregate tissue pieces by 10 20 strokes Transfer homogenized mixture to a 15 ml conical tube and centrifuge at 3000 rpm for 5 min 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 min at 4 C Then go directly to Step 3g 3 Cell Lysis and Chromatin Extraction Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Mix and centrifuge at 1000 rpm for 5 min Remove medium and wash cells once with 10 ml of ice cold PBS by centrifuging at 1000 rpm for 5 min Add Working Lysis Buffer to re suspend the cell pellet 200 pl 1x10 cells for adherent cells and 100 ul 1x10 cells for suspension cells Transfer cell suspension to a 1 5 ml vial and incubate on ice for 10 min Vortex vigorously for 10 sec and centrifuge at 5000 rpm for 5 min Carefully remove supernatant Add Working Extraction Buffer to re suspend
10. istone H3K9 Monomethy Polyclonal Antibody A 4035 Histone H3K9 Dimethyl Polyclonal Antibody A 4036 Histone H3K9 Trimethy Polyclonal Antibody A 4037 Histone H3K27 Monomethyl Polyclonal Antibody A 4038 Histone H3K27 Dimethyl Polyclonal Antibody A 4039 Histone H3K27 Trimethy Polyclonal Antibody A 4040 Histone H3K36 Monomethy Polyclonal Antibody A 4041 Histone H3K36 Dimethyl Polyclonal Antibody A 4042 Histone H3K36 Trimethyl Polyclonal Antibody A 4043 Histone H3K79 Monomethy Polyclonal Antibody A 4044 Histone H3K79 Dimethyl Polyclonal Antibody A 4045 Histone H3K79 Trimethy Polyclonal Antibody A 4046 Histone H4K20 Monomethy Polyclonal Antibody A 4047 Histone H4K20 Dimethyl Polyclonal Antibody A 4048 Histone H4K20 Trimethy Polyclonal Antibody A 4049 Phospho Histone H3 Ser10 Monoclonal Antibody A 4050 Phospho Histone H3 Ser28 Polyclonal Antibody Page 8 Printed 2014 09 22 110 Bi County Blvd Ste 122 Farmingdale NY 11735 P 2001 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
11. omatin including ChIP in vitro protein DNA interaction analysis nuclear enzyme assay etc PRINCIPLE amp PROCEDURE The ChromaFlash Chromatin Extraction Kit contains all reagents required for carrying out successful chromatin extraction directly from mammalian cells or tissues Cell membranes of the sample with or without cross linking are broken down using the provided lysis buffer Chromatin or DNA protein complex is then extracted with the extraction buffer The extracted chromatin can then be diluted with chromatin buffer and stored at the appropriate temperature Page 3 Printed 2014 09 22 110 Bi County Blvd Ste 122 Farmingdale NY 11735 P 2001 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 Start with cell or tissue sample with or without cross linking Lyse sample with lysis buffer D Normal mouse IgG O RN Apolymerase Il with extraction buffer Relative Enrichment Fold gt Obtain isolated chromatin GAPDH MLH 1 26 L Extract chromatin F ChIP analysis of RNA polymerase Il enriched in GAPDH and MLH1 promoters with chromatin extract Schematic procedure of the ChromaFlash prepared from formaldehyde fixed colon cancer cells Chromatin Extraction Kit 2x1 0 using the ChromaFlash Chromatin Extraction Kit ASSAY PROTOCOL For the best results please read
12. the protocol in its entirety prior to starting your experiment Starting Materials Monolayer cells 1x10 to 5x10 cells per preparation Suspension cells 1x10 to 5x10 cells per preparation Tissues 10 mg to 200 mg per preparation 1 Preparation of Working Buffers and Solutions a Prepare Working Lysis Buffer by adding 1 ml of 10X Lysis Buffer and 6 ul of Protease Inhibitor Cocktail to every 9 ml of distilled water b Prepare Working Extraction Buffer by adding 1 ul of Protease Inhibitor Cocktail to every 1 ml of Extraction Buffer Page 4 Printed 2014 09 22 110 Bi County Blvd Ste 122 Farmingdale NY 11735 P 2001 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 2 Cell Collection and Cross Linking 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 For Monolayer or Adherent Cells Grow cells treated or untreated to 80 90 confluence on a 100 mm plate then trypsinize and collect them into a 15 ml conical tube Count the cells in a hemocytometer Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant Wash cells with 10 ml of PBS once by centrifugation at 1000 rpm for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3d after Step 2c
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