Pre-Sure™ ChIP Antibody Validation Kit
Contents
1. Pre Sure ChIP Antibody Validation Kit Base Catalog P 2031 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The Pre Sure ChIP Antibody Validation Kit is used for determining if antibodies are qualified for chromatin immunoprecipitation ChIP using chromatin isolated from various species particularly mammals in a high throughput format Chromatin can be isolated and sheared using your own successful method or commercially available kit For your convenience and the best results Epigentek s ChromaFlash Chromatin Isolation amp Shearing Kit Cat P 2023 is optimized for use with this product Input Amount of Chromatin The amount of chromatin for each reaction can be 0 2 ug about 2 x 104 cells to 5 ug about 0 5 x 10 cells depending on whether the antibody targeting proteins are highly abundant or not For an optimal reaction the input chromatin amount should be 1 ug about 1 x 10 cells since enrichment of target proteins to genome loci varies and some target proteins are of low abundance Starting Materials Starting materials can include various tissue or cell samples such as cultured cells from a flask or dish Antibodies The antibodies to be tested can be monoclonal and polyclonal antibodies generated from any species and should be IgG type For the best results the specificity of the antibodies should be pre tested by ELISA or Western blot Internal Controls The assay standard negative non immune IgG con2. 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 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 Sheared chromatin t Protein DNA complex antibody IP Add fluoro assay solution after wash v Signal measurement Fig 1 Schematic procedure of the Pre Sure ChIP Antibody Validation Kit ASSAY PROTOCOL 500 450 400 R 0 9366 350 300 250 200 150 100 qPCR GAPDH Amplification fold 50 0 5 10 15 20 25 ChIP Grade Intensity fold Fig 2 ChiP grade Antibody Validation Core chromatin isolated from Hela cells was used for measuring ChIP Grade Intensity CGI of each antibody using the Pre Sure ChIP Antibody Validation Kit To confirm the CGI results ChIP qPCR analysis was performed using the same antibodies to enrich the protein or modified histone DNA complexes on euchromatin A euchromatic GAPDH locus served as a reference The antibodies H3K4m2 3 H3K9ac 4 Dnmt1 5 H3K18ac 6 and H3K14ac 7 which are demonstrated to be ChIP grade by epigenetics researchers highly enriched the proteins modified his tones associated with euchromatin while antibodies HDAC4 1 and H3K9m2 2 which are less associated with euchromatin show no little enrichment For the best results please read the protocol in its entirety prior to startin
3. Web www epigentek com Printed 2014 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 Currently there are no assays or tests available to quickly check if the target protein DNA complex was successfully enriched by the antibody unless a downstream application such as PCR microarray or sequencing is performed Routinely validating if the target protein DNA complex is successfully enriched by the antibody before downstream application would ensure the success of a ChIP and allow time saving labor reduction and cost effectiveness As the leading provider of various ChIP kits and epigenetic antibodies Epigentek utilized its expertise to develop the Pre Sure ChIP Antibody Validation Kit the first commercially available product for fast validation of ChlIP grade antibodies The Pre Sure ChIP Antibody Validation Kit has the following advantages e High Speed The entire procedure can be completed in less than 3 hours with the actual handling time being less than 30 minutes e High Specificity Detected signals are specifically from the target protein DNA complex captured by the antibody and fully consistent with results obtained by ChIP qPCR e High Efficiency Direct quantification of target protein DNA complexes captured by the antibody No need for PCR amplification and sequencing e High Sensitivity As low as 0 5 ng of the enriched target protein DNA complex can be detected e High Through
4. or over cross linking The optimal amount of chromatin per ChIP reaction is 0 2 5 Lg which may be obtained from 0 5 1x10 cells respectively The minimal amount of chromatin that should be used is 0 2 ug which may be obtained from 20 000 cells Appropriate chromatin cross linking is also required Insufficient or over crosslinking will cause dissociation of the target protein from DNA or less antibody binding During the cross linking step of chromatin preparation ensure that the cross linking time is from 10 15 min the final concentration of formaldehyde is 1 and or the quench solution is 0 125 M glycine Improper antibody binding or ChIP reaction conditions with antibody Check if the antibody binding and ChIP reaction conditions and steps are correct Inappropriate chromatin shearing conditions If chromatin is from specific cell tissue types or is differently fixed the shearing conditions should be optimized to allow the DNA fragment size to be between 250 1000 bps 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 07 02 P 2031 Little or No Signal from Sample Positive Control 1 and Positive Control 2 Wells Incorrect temperature and or insufficient time during antibody bindin
5. prepared chromatin can be directly used for the reaction Frozen chromatin samples should be thawed quickly at RT and then placed on ice before use Store remaining chromatin samples at 20 TC or at 80 C if they will be not used within 8 hours d Cap wells with Adhesive Covering Film strips and incubate at room temperature for 90 min on an orbital shaker 100 rpm For low abundance targets the incubation time should be extended to 2 3 hours 4 Washing of the Reaction Wells a Carefully remove the solution from each well and discard it b Wash each well four times with 200 ul of Diluted WB1 each time To wash the wells pipette Diluted WB1 into the wells then remove it from the wells and discard it Repeat four a total of four washes c Wash each well one time with 200 ul of WB2 To wash the wells pipette WB2 into the wells and then remove it from the wells and discard it 5 Signal Detection and Calculation a Prepare the 1X Fluoro Assay Solution by adding 1 ul of 100X Fluoro Assay Solution into 99 ul of WB2 b Add 100 ul of 1X Fluoro Assay Solution into each well and incubate at room temperature for 1 3 minutes away from light Measure and read fluorescence on a fluorescence microplate reader at 490 ex 530em nm The signal is stable for about 2 hours c Calculate ChIP grade intensity of antibody using the following formula Antibody RFU Blank RFU ChIP Grade Intensity CGI IgG RFU Blank RFU 110 Bi County Bl
6. d incubate the wells at room temperature for 60 min 3 Preparation of ChIP Reaction a Carefully peel away the Adhesive Covering Film on the antibody binding wells from Step 2 to avoid contamination between 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 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 io co on A se WE b Using a pipette remove the antibody reaction solution and Non immune IgG solution from each well and wash the wells one time with 150 ul of CB ChIP Buffer To wash the wells pipette CB ChIP Buffer into the wells then remove it from the wells and discard it c Setup the ChIP reactions by adding the reagents to the wells that are bound with antibodies sample and positive control wells or IgG negative control well according to the following chart Positive Positive Negative Negative FECES Seo pani Control 1 Control 2 Control 1 Control 2 CB ChIP Buffer 90 98 ul 100 ul 90 98 ul 97 5 ul 90 98 ul 97 5 ul Chromatin 2 t10w Oo 240w o 20w o0 Positive Core Chromatin 0 0 0 2 5 ul 0 2 5 ul Note The final amount of chromatin should be 1 ug well 1 X 10 cells may yield 1 ug of chromatin Sonicated chromatin can be further diluted with CB ChIP Buffer to desired concentration Freshly
7. g and ChIP reaction Ensure the incubation times and temperatures described in the protocol are followed correctly Improper measurement of signals Ensure the proper wavelength 490ex 530em nm is set Improper sample or kit storage Chromatin sample should be stored at 80 C for no longer than 6 months preferably less than 3 months Avoid repeated freeze thaw cycles Each component of the kit should be stored according to the user guide No Difference in Signal Intensity Between Negative and Positive Control Wells Insufficient washing Check if washing recommendations at each step is performed according to the protocol If the signal intensity in the negative control is still high washing stringency can be increased in the following ways 1 Increase wash time at each wash step after adding Diluted WB1 leave it in the wells for 2 3 min and then remove it 2 Add an additional one to two washes The provided volume of WB1 is sufficient for 4 extra washes for each sample Little or No Signals Generated From Each Sample Well Only Insufficient amount of the antibodies added into the reaction Check if the amount of antibody added into the reaction is sufficient 0 6 ug well and if not increase the amount of antibody RELATED PRODUCTS ChIP Reaction P 2002 P 2003 P 2014 P 2025 P 2026 EpiQuik Chromatin Immunoprecipitation ChIP Kit EpiQuik Tissue Chromati
8. g your experiment Starting Materials Input Chromatin Amount Chromatin amount can range from 0 2 ug to 5 ug per reaction An optimal amount is 1 ug per reaction Chromatin isolation shearing You can use your method of choice for chromatin isolation and shearing Epigentek offers the ChromaFlash Chromatin Isolation Shearing Kit Cat P 2023 for your convenience Chromatin can be isolated from various tissue and cells For the best results the chromatin should be isolated from cultured cell lines grown in log phase For histone targeting 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 Page 5 Printed 2014 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 io co on A se WE antibodies the tissue cell cross linking is not required However for non histone targeting antibodies such as those for transcription factors the tissue cells should be cross linked before chromatin isolation The chromatin can be sheared by water bath based sonication or probe based sonication according to the supplier s instructions The sheared DNA chromatin size should be between 150 1000 bps Chromatin Storage Isolated chromatin can be stored at 20 C short term or 80 C long term until use 1 Prepare Diluted WB1 1X Wash Buffer 48 Assay Kit Add 13 ml of WB1 10X Wash Buffer to 117 ml of dis
9. he 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 Pre Sure ChIP Antibody Validation Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The Pre Sure ChIP Antibody Validation Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW The interactions between protein and DNA plays 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 interactions is important for understanding cellular process Chromatin immunoprecipitation ChIP
10. n Immunoprecipitation ChIP Kit EpiQuik Plant ChIP Kit ChromaFlash One Step ChIP Kit ChromaFlash One Step Magnetic ChIP Kit Chromatin Preparation P 2001 P 2023 ChromaFlash Chromatin Extraction Kit ChromaFlash Chromatin Isolation amp Shearing Kit Sonication Instruments EQC 1100 PCR Analysis P 1029 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 EpiSonic Multi Functional Bioprocessor 1100 EpiQuik Quantitative PCR Fast Kit Page 9 Printed 2014 07 02 P 2031
11. offers an advantageous tool for studying protein DNA interactions It detects that a target protein binds to specific sequences of a gene in living cells by PCR ChIP PCR microarrays ChIP chip or sequencing ChIP seq In particular ChIP that uses antibodies directly against various transcription factors TF for genome wide transcription factor binding site analysis by next generation sequencing is in high demand Reliable enrichment of target protein DNA complexes is critical for deriving high quality data from ChIP based PCR microarray and sequencing More importantly the qualified ChIP antibody determines the success or failure of a ChIP There are many antibodies against the same protein antigen target available today The affinity and specificity of these antibodies are generally determined using complementary methods such as Western blot immunofluorescence peptide arrays and peptide competition assays However most of these antibodies are not validated for their ability to enrich target protein DNA complexes contained in chromatin Furthermore some antibodies labeled as ChIP validated available on the market may not be internally tested Even if the published data shows that it is a ChIP validated antibody the same results may not always be reproduced due to Lot to Lot variation of the antibody 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
12. om before 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 Positive Control and Fluoro Assay Solution at 20 C 2 Store WB1 Non Immune IgG AcH3 Antibody and 8 Well Assay Strips With 1 Frame at 4 C away from light 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 Check if WB1 contains salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until the salts are re dissolved MATERIALS REQUIRED BUT NOT SUPPLIED Incubator oven with variable temperature Adjustable pipette and multiple channel pipette Aerosol resistant pipette tips Antibodies of interest Chromatin extracts Oo Oad00 oO Orbital shaker 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 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 O Fluorometer Fluorescence microplate reader O Solution reservoirs GENERAL PRODUCT INFORMATION Quality Control Each lot of the Pre Sure ChIP Antibody Validation Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees t
13. put 96 strip well plate format allow for validation of a single antibody each time or up to 43 antibodies simultaneously e High Convenience An assay control core chromatin negative control non immune IgG and positive control antibody are provided in the kit and are used for determination of ChIP enrichment efficiency specificity of the validation test whether test was successfully performed overall PRINCIPLE amp PROCEDURE The Pre Sure ChIP Antibody Validation Kit contains all necessary reagents required for carrying out a successful ChIP antibody validation test using sheared chromatin from mammalian cells or tissue This kit includes a positive control antibody a negative control non immune IgG and core chromatin extract that can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol The antibodies are first bound to the assay wells The positive control antibody AcH3 is ChIP grade which can immunoprecipitate the acetylated histone H3 complexed with DNA in core chromatin and generate a fluorescent signal in the reaction with the Fluoro Assay Solution Similarly if the tested antibody works in ChIP it can also capture target protein DNA complex from chromatin and generate a fluorescent signal The signal intensity is proportional to the amount of protein DNA complexes captured by the antibody and accordingly indicates if the antibody is ChIP grade and the grade intensity 110 Bi County Blvd
14. tilled water 96 Assay Kit Add 26 ml of WB1 10X Wash Buffer to 234 ml of distilled water This Diluted WB1 1X Wash Buffer can now be stored at 4 C for up to six months 2 Antibody Binding to Strip Wells a Predetermine the number of strip wells required for your experiment 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 Setup the antibody binding reactions by adding the reagents to each well according to the following chart Positive Controls Negative Controls Reagents Samples 1 and 2 1 and 2 AB Antibody Buffer 100 ul 100 ul 100 ul 100 ul Your Antibodies 0 5 2 ul 0 0 0 AcH3 Antibody 0 0 6 ul 0 0 Non Immune IgG 0 0 0 6 ul 0 Note The final amount of each component should be a antibodies of interest 0 6 ug well b ACH3 antibody 0 6 ug well and c non immune IgG 0 6 ug well The final volume in each assay well can range from 100 5 ul to 102 ul This slight variation in volume for each well will not affect the assay performance The volume in each assay well should not exceed 102 ul Two positive and two negative controls should be set up for the assay One positive and one negative control for the input chromatin sample and one positive and one negative control for the provided Positive Control Core Chromatin and Non lmmune IgG respectively Seal the wells with Adhesive Covering Film strips an
15. trol and positive antibody control are provided in this kit The assay standard is used to determine ChIP enrichment efficiency and the negative and positive controls can be used for determining the specificity of the validation test and if the test was successfully performed 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 Farmingdale 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 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 KIT CONTENTS 48 assays 96 assays Storage Components P 2031 48 P 2031 96 Upon Receipt WB1 10X Wash Buffer 1 15 ml 30 ml 47 WB2 Wash Buffer 2 15 ml 30 ml RT AB Antibody Buffer 6 ml 12 ml RT CB ChIP Buffer 15 ml 30 ml RT Positive Control Core Chromatin 400 ug ml 20 ul 40 ul 20 C Non Immune IgG 1 mg ml 10 ul 20 ul 47 AcH3 Antibody 0 6 mg ml 5 ul 10 ul 4 Fluoro Assay Solution 100 X 60 ul 120 ul 20 C 8 Well Assay Sirips With 1 Frame 6 12 4 Adhesive Covering Film 1 2 RT User Guide 1 1 RT Spin the solution down to the bott
16. vd 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 07 02 Epigentek Group Inc All rights reserved Products are for research use only P 2031 In general an antibody is considered ChIP grade if the CGI is gt 4 A greater CGI indicates an antibody which is more robust for ChIP Antibodies that are validated using this assay can be further confirmed by performing a proper ChIP gPCR test SUGGESTED STRIP WELL SETUP The suggested strip well plate setup in a 48 assay format for 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 Negative Control 1 Negative Control 1 Sample Sample Sample Sample c Negative Control 2 Negative Control 2 Sample Sample Sample Sample D Positive Control 1 Positive Control 1 Sample Sample Sample Sample E Positive Control 2 Positive Control 2 Sample Sample Sample Sample F Sample Sample Sample Sample Sample Sample G Sample Sample Sample Sample Sample Sample H Sample Sample Sample Sample Sample Sample TROUBLESHOOTING Problem Possible Cause Suggestion Little or No Signal from both Sample and Positive Control 1 Wells Poor chromatin quality due to insufficient amount of cells or insufficient
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