ChromaFlash High-Sensitivity ChIP Kit
Contents
1. b Setup the antibody binding reactions by adding the reagents to each well according to the following chart Reagents Sample Positive Control Negative Control AB Antibody Buffer 50 80 ul 50 80 ul 50 80 ul Your Antbodies O52u o v AMENA Polymerasel 0 C Norimmune 6 o o 08y Note The final amount of each component should be a antibodies of interest 0 8 ug well b RNA Polymerase II 0 8 ug vell and c non immune IgG 0 8 ug vell The amounts of the positive control Anti RNA Polymerase Il and negative control Non lmmune IgG are sufficient for matched use wth samples if two antibodies are used for each sample or one antibody is used for two of the same samples If using one antibody of interest for each sample wth matched use of the positive and negative control extra RNA Polymerase Il Non lImmune IgG and 8 Well Strips required can be separately obtained from Epigentek c Seal the wells with Adhesive Covering Film Strips and incubate the wells at room temperature for 60 90 min on an orbital shaker 100 rpm Meanwhile perform the steps from Section 3 Cell Collection and Cross Linking to Section 5 Chromatin Shearing 3 Cell Collection and Cross Linking 3 1 For Monolayer or Adherent Cells a Grow cells treated or untreated to 80 90 confluence on a 6 well plate or 100 mm dish the number of cultured MDA 231 cancer cells on an 80 90 confluent plate is listed in the table below as 110 Bi Cou2. ChromaFlash High Sensitivity ChIP Kit Base Catalog P 2027 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The ChromaFlash High Sensitivity ChIP Kit is suitable for selective enrichment of a chromatin fraction containing specific DNA sequences which is in a high throughput format with high sensitivity and specificity using various mammalian cell tissues The optimized protocol and kit components reduce non specific background ChIP levels to allow capture of low abundance protein transcription factors and increased specific enrichment of target protein DNA complexes The target protein bound DNA prepared with the ChromaFlash High Sensitivity ChIP Kit can be used for various downstream applications including PCR ChIP PCR microarrays ChIP on chip and sequencing ChIP seq Input Amount of Tissue Cells In general the amount of cells and tissues for each reaction can be 2 x 10 to 1 x 10 and 0 5 mg to 50 mg respectively For optimal preparation the input amount should be 1 to 2 x 10 cells or 10 to 20 mg tissues since the enrichment of target proteins to genome loci may vary For the target proteins that are low abundance transcription factors the input amount should be 5 to 6 x 10 cells or 50 60 mg tissues Starting Materials Starting materials can include various tissue or cell samples such as cells from flask or plate cultured cells fresh and frozen tissues etc Antibodies Antibodies should be ChIP or IP grade i
3. the binding site of the protein of interest Chromatin sample should be stored at 80 C for no longer than 6 months preferably less than 3 months Avoid repeated freeze thaw cycles DNA samples should be stored at 20 C for no longer than 6 months preferably less than 3 months Check if washing recommendations at each stepis performed according to the protocol If the signal intensityin the negative control is still high washing stringencycan be increased in the following ways 1 Increase wash time ateach wash step after adding WB leave itin the wells for 3 4 min and then remove it 2 Add an additional one wash with WB respectively the provided volume of WBis sufficientfor 4 extra washes for each sample Decrease the number of PCR cycles i e 32 35 cycles to keep amplification at the exponential phase This will reduce high background in endpoint PCR and allow differences in amplification to be seen Realtime PCR is another choice in suchcases Increase the antibody amountand use ChIP grade antibodies validated for use in ChIP 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 15 Printed 2015 07 30 P 2027 PCR primers are not optimized Confirm species specificity of primers Primers should be designed to cover
4. 10 mg ml 120 ul RNase A 10 mg ml 60 ul GAPDH Primer Forward 20 uM 16 pl GAPDH Primer Reverse 20 uM 16 ul 8 Well Assay Strips With 1 Frame 8 Well Strip Caps Adhesive Covering Film Pt 2 F Spin Column 50 3 50 1 F Collection Tube 300 Spin the solution down to the bottom prior to use 1 0 0 1 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 Enrichment Enhancer and RNase A at 20 C Store WB AB CB BS Protease Inhibitor Cocktail Non Immune IgG Anti RNA Polymerase II Proteinase K GAPDH Primer Forward GADPH Primer Reverse and 8 Well Assay Strips With 1 Frame at 4 C away from light 2 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 WB and CB contain salt precipitates before use If so briefly warm at room temperature or 37 C and shake the buffer until the salts are re dissolved 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 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 MATERIALS REQUIRED BUT NOT SUPPLIED Sonicator Vortex mixer Dounce homogenizer with
5. Bi County Blvd Ste 122 Farmingdale NY 11735 Page 11 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed pincer Epigentek Group Inc All rights reserved Products are for research use only P 2027 b Add 40 ul of DRB RNase A to each well and then cover with Strip Caps c Incubate the wells at 42 C for 30 min d Add 2 ul of Proteinase K to each of the wells and re cap the wells e Incubate the wells at 60 C for 45 min f Quickly transfer the DNA solution from each well to 0 2 ml strip PCR tubes Cap the PCR tubes g Incubate the PCR tubes containing DNA solution at 95 C for 15 min in a thermolcycler h Place the PCR tubes at room temperature If liquid is collected on the inside of the caps briefly spin the liquid down to the bottom DNA prepared at this step can be directly used for PCR or go to Step 8 i for further purification for use in ChIP Seq i Place a spin column into a 2 ml collection tube Add 200 ul of DBS DNA Binding Solution to the samples and transfer the mixed solution to the column Centrifuge at 12 000 rom for 30 seconds j Add 200 ul of 90 ethanol to the column centrifuge at 12 000 rpm for 30 seconds Remove the column from the collection tube and discard the flowthrough k Replace column to the collection tube Add 200 ul of 90 ethanol to the column and centrifuge at 12 000 rpm for 30 seconds Remove the column and discard the flowthrou
6. a short sequence region 70 150 bp for more efficientand precise amplification of target DNA region the binding site of the protein of interest RELATED PRODUCTS ChIP Reaction P 2002 EpiQuik Chromatin Immunoprecipitation Kit P 2003 EpiQuik Tissue Chromatin Immunoprecipitation ChIP Kit P 2014 EpiQuik Plant ChIP Kit P 2025 ChromaFlash One Step ChIP Kit P 2026 ChromaFlash Magnetic One Step ChIP Kit Chromatin Preparation and Cleanup P 2001 ChromaFlash Chromatin Extraction Kit P 2023 ChromaFlash Chromatin Isolation amp Shearing Kit P 1006 DNA Concentrator Kit Sonication Instruments EQC 1100 EpiSonic Multi Functional Bioprocessor 1100 PCR Analysis P 1029 EpiQuik Quantitative PCR Fast Kit DNA Library Prep P 1051 EpiNext DNA Library Preparation Kit Illumina For ChIP grade antibodies please search chip grade at wwwv epigentek com 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 16 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027
7. on ice between each pulse The conditions of cross linked DNA shearing can be optimized based on cells and sonicator equipment Note When probe based sonication is carried out the shearing effect may be reduced if foam is formed in chromatin sample solution Under this condition discontinue sonication and centrifuge the sample at 4 C at 12 000 rpm for 3 min to remove the air bubbles The isolated chromatin can also be sheared wth various enzyme based methods Optimization of the shearing conditions for example enzyme concentration and incubation time is needed in order to use enzyme based methods Centrifuge at 12 000 rpm at 4 C for 10 min after shearing Transfer supernatant to a new wal The chromatin solution can now be used immediately or stored at 80 C after aliquoting appropriately until further use Avoid multiple freeze thaw cycles Note The size of sonicated chromatin should be verified before starting the immunoprecipitation step The following steps can be carried out to isolate DNA for gel analysis of DNA fragment size 1 add 25 tl of each chromatin sample to a 0 2 mI PCR tube followed by adding 25 ul of DRB DNA Release Buffer and 2 ul of Proteinase K 2 incubate the sample at 60 for 30min followed by incubating at 95 for 10 min 3 spin the solution down to the bottom 4 transfer supernatant to anew 0 2 mI PCR vial Use 30 40 ul for DNA fragment size analysis along wth a DNA marker on a 1 2 agarose gel a
8. pl Final Concentration EpiQuik Master Mix 2X 10 ul ZEE DNA Template 50 pg 0 1 ug DNA RNA Free H20 Total Volume For the negative control use DNA RNA free water instead of DNA template 0 40 5 uM Program the PCR Reactions Place the reaction plate in the instrument and set the PCR conditions as follows Cycle Step Cyce 95 C 10 sec Cycling 55 C 10sec 40 72 C 8 sec FinalEviension Fold Enrichment Calculation Fold enrichment FE can be calculated by simply using a ratio of amplification efficiency of the ChIP sample over that of Non Immune IgG Amplification efficiency of RNA Polymerase II can be used as a positive control FE 2196 CT Sample CT x 100 For example if Ct for IgG is 38 and the sample is 34 then 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 13 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 FE 28 x100 1600 Endpoint PCR Primer Design The primers designed should meet the criteria for endpoint PCR For example the covered sequence region should be 100 400 bp in length PCR primer design tools e g Primer3Plus can be used to help in the selection of appropriate primer pairs PCR Reaction Endpoint PCR can be performed using your own proven method It is important to stop the PCR rea
9. R assay while the recruitment of meH3 K9 to the promoters on a genome wide scale can be detected by ChIP chip or ChiP sequencing 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 Such analysis requires that ChlPed DNA contain minimal background for reliably identifying true TF enriched regions High background in ChIP is mainly caused by the following weaknesses ineffective wash buffer insufficient cross link reversal inappropriate DNA fragment length and residual RNA interference Thus for effectively capturing TF DNA complexes which are often in low abundance an ideal ChIP method requires having maximum sensitivity with minimized background levels This method should also be able to enrich highly abundant protein DNA complexes using a small amount of cells or tissues in a high throughput format Epigentek s ChromaFlash High Sensitivity ChIP Kit is designed to achieve these goals by maximizing sensitivity and minimizing non specific background signals The ChromaFlash High Sensitivity ChIP Kit has the following advantages e Optimized buffers and protocol allow minimal ChIP background by overcoming the weaknesses that cause non specific enrichment thereby increasing sensitivity and specificity of the ChIP reaction e Increased antibody selectivity and capture efficiency through the use
10. and 50 80 ul of CB ChIP Buffer can be used For lowabundance targets 2 ul of Enrichment Enhancer and 88 ul of chromatin can be used without adding CB ChIP Buffer Freshly 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 C or 80 if they will be not used within 8 hours Input DNA control is only used for estimating the enrichment efficiency of ChIP and is generally not necessary since the positive and negative control can be used for estimating the same objective more accurately If you would like to include the input DNA control the purified inout DNA prepared at Step 5d Note can be used Cap wells with Strip Caps and incubate at room temperature for 60 90 min on an orbital shaker 100 rpm For low abundance targets the incubation time should extend to 2 3 hours or at 4 C overnight 7 Washing of the Reaction Wells a b C Carefully remove the solution and discard from each well Wash each well with 200 ul of the WB each time for a total of 4 washes Allow 2 minutes on an orbital shaker 100 rpm for each wash Pipette wash buffer out from the wells Wash each well with 200 ul of the DRB one time by pipetting DRB in and out of the well 8 Reversal of Cross Links Release and Purification of DNA a Prepare RNase A solution by adding 1 ul of RNase A to 40 ul of DRB 110
11. ated for high abundance targets from a single ChIP reaction well However this may be difficult for low abundance target enrichment For lowabundance targets we recommend pooling the DNA solution from several wells with the same ChIP reaction to gain 10 ng or more DNA amount We also recommend performing qPCR to verify the quality of the ChiPed DNA 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 12 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 PCR ANALYSIS Real Time PCR Primer Design The primers designed should meet the criteria for real time PCR For example the covered sequence region should be 50 150 bp in length G C stretches at 3 ends of primers should be avoided PCR Reaction Real time PCR can be performed using your own proven method For your convenience and the best results Epigentek offers the EpiQuik Quantitative PCR Fast Kit Cat No P 1029 which is optimized for fast GPCR reactions As an example the protocol is presented below Prepare the PCR Reactions Thaw all reaction components including master mix DNA RNA free water primer solution and DNA template Mix well by vortexing briefly Keep components on ice while in use and return to 20 C immediately following use Add components into each well according to the following Component Size
12. bate on ice for 10 min Note If the total solution volume is less than 1 5 ml transfer the solution to a 1 5 ml microtube Vortex vigorously for 10 sec and centrifuge at 3000 rpm for 5 min Then go to Step 4a For Suspension Cells Collect cells treated or untreated into a 15 ml conical tube 2 x10 to 5x10 cells are required for each ChIP reaction Count cells ina 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 rom for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3 2 i after Step 3 2 c Add 9 ml fresh cell culture medium containing formaldehyde to 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 Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution Page 8 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 3 3 a b 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 Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only Mi
13. centration of formaldehyde is 1 and the quench solutionis 0 125M glycine Increase the antibody amountand use ChIP grade antibodies validated for use in ChIP If chromatin is from a specific cell tis sue type or is differently fixed the shearing conditions should be optimized to allow DNA fragmentsize to be between 200 800 bp Ensure the incubation times and temperatures described in the protocol are followed correctly Page 14 Printed 2015 07 30 P 2027 No difference in signal intensitybetween negative and positive control wells Little or no PCR products generated from sample wells only Improper PCR conditions including improper PCR programming PCR reaction solutions and or primers Improper sample storage Insufficientwashing Too many PCR cycles Plateau phase ofamplification caused by excessive number of PCR cycles in endpoint PCR may mask the difference of signal intensity between negative contol and positive control Poor enrichmentwith antibody Some antibodies used in ChIP mightnot efficiently recognize fixed protein Ensure the PCR is properly programmed If using ahomebrew PCR reaction solution check if each componentis correctly mixed If using a PCR commercial kit check if it is suitable for your PCR Confirm species specificity of primers Primers should be designed to cover a shortsequence region 70 150 bp for more efficientand precise amplification of target DNA region
14. ction at the exponential phase by setting up an appropriate number of PCR cycles in order to make a reliable comparision of enrichment efficiency obtained from different ChIP reactions Thus the optimized number of PCR cycles should be determined empirically PCR Product Analysis Endpoint PCR products can be analyzed by separating amplicons on a 1 2 agarose gel followed by staining with ethidium bromide and visulizing with UV illumination TROUBLESHOOTING Little or no PCR products generated from both sample and positive control wells Poor chromatin quality due to insufficientamountofcells or insufficient or over cross linking Poor enrichmentwith antibody some antibodies used in ChIP mightnot efficiently recognize fixed protein Inappropriate DNA fragmenting condition Incorrect temperature and or insufficienttime during DNA release 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 optimal amountofchromatin obtained per ChIP reaction should be 2 4 ug about 2 4 x 10 cells Appropriate chromatin cross linking is also required Insufficient or over crosslinking will cause DNAloss or increased background During the cross linking step of chromatin preparation ensure thatthe cross linking time is within 10 15 min the final con
15. ection 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 High Sensitivity ChIP Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The ChromaFlash High Sensitivity ChIP Kit and methods of use contain proprietary technologies by Epigentek A BRIEF OVERVIEW Protein DNA interaction 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 interaction is important for understanding cellular processes Chromatin immunoprecipitation ChIP offers an advantageous tool for studying protein DNA interactions It allows for the detection of a specific protein bound to a specific gene sequence in living cells using PCR ChIP PCR microarrays ChIP chip or sequencing ChIP seq For example 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 PC
16. gh Replace column to the collection tube and wash the column again with 200 ul of 90 ethanol at 12 000 rpm for 1 min m Place the column in a new 1 5 ml vial Add 20 ul of DEB DNA Elution Buffer directly to the filter in the column and centrifuge at 12 000 rpm for 30 seconds to elute the purified DNA Purified DNA is now ready for PCR ChIP chip and ChIP seq use or storage at 20 C Note For real time PCR analysis we recommend the use of 1 2 ul of eluted DNA in a 20 ul PCR reaction If input DNA wil be used it should be diluted 10 fold before adding to the PCR reaction Control primers 110 bp for human cells included in the kit can be used as a positive control For end point PCR the number of PCR cycles may need to be optimized for better PCR results In general the amplification difference between normal IgG control and positive control may vary from 3 to 8 cycles depending on experimental conditions For ChIP Seq if sequencing is on MiSeq HiSeq Hiscan SQ or Genome Analyzer ChIP DNA library can be constructed using our EpiNext DNA Library Preparation Universal Kit Illumina Cat P 1051 or Illumina ChIP seq DNA Sample Prep Kit For sequencing on other sequencing platforms appropriate library preparation methods that are compatible wth these platforms should be selected accordingly In general at least 10 ng of ChilPed DNA is required for ChIP DNA library construction This amount can easily be gener
17. n order to recognize fixed and native proteins that are bound to DNA or other proteins If you are using antibodies which have not been validated for ChIP then appropriate control antibodies such as RNA Polymerase Il Cat A 2032 should be used to demonstrate that the antibody and prepared chromatin are suitable for ChIP Internal Controls Both negative and positive ChIP controls are provided in this kit Precautions To avoid cross contamination carefully pipette the sample or solution into the tube 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 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 KIT CONTENTS Component 48 reactions Cat P 2027 24 Cat P 2027 48 Upon Receipt WB Wash Buffer 2x25ml AB Antibody Buffer 6 ml LB Lysis Buffer 28 ml CB ChIP Buffer 12 ml DRB DNA Release Buffer 16 ml DBS DNA Binding Solution 14 ml BS Blocker Solution 4ml 4 DEB DNA Elution Buffer 2ml Enrichment Enhancer 110 ul Protease Inhibitor Cocktail PIC 60 ul Non Immune IgG 1 mg ml 20 ul Anti RNA Polymerase Il 1 mg ml 16 ul Proteinase K
18. nd 5 Stain wth ethidium bromide or other fluorescent dye for DNA and visualize it under ultraviolet light The length of sheared DNA should be between 100 700 bps wth a peak size of 300 bps 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 m Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 6 Preparation of ChIP Reaction a Carefully peel away the Adhesive Covering Film on the antibody binding wells from Step 2b to avoid contamination between each well 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 Set up 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 Reagents Sample Positive Control Negative Control CB ChIP Buffer 48 78 l 48 78 ul 48 78 ul 10 40 pi 10 40 i 10 40 pi Enrichment Enhancer BS Blocker Solution 10 ul 10 ul 10 ul 7004 100 700 Note The final amount of chromatin should be 2 yg well 2 X 10 cells may yield 1 ug of chromatin Sonicated chromatin can be further diluted wth CB ChIP Buffer to desired concentration For histone samples wth sufficient chromatin gt 0 5 ug the Enrichment Enhancer is not required
19. ntitative PCR Fast Kit Cat No P 1029 10000 m Non Immune IgG 1000 4 Z w RNA polymerase Il antibody LL D 100 xe a W o 10 2 amp c9 x aT 0 2 000 4 000 20 000 100 000 500 000 Cell Number Fig 3 High abundance protein enrichment Sheared chromatin isolated from different numbers of MBD 231 cells was used for ChIP qPCR analysis of RNA polymerase Il enrichment in GAPDH promoters using the ChromaFlash Hi Sensitivity ChIP Kit Cat No P 2027 and EpiQuik Quantitative PCR Fast Kit Cat No P 1029 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 Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 ASSAY PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment 1 Preparation of Working Buffers and Solutions a Prepare Working Lysis Buffer by adding 6 ul of Protease Inhibitor Cocktail to every 10 ml of LB Lysis Buffer b Prepare Working CB ChIP buffer by adding 1 ul of Protease Inhibitor Cocktail to every 1 ml of CB ChIP Buffer 2 Antibody Binding to Assay Strip Wells a Predetermine the number of Assay Strip Wells required for your experiment Carefully remove any unneeded strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C
20. nty 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 Web www epigentek com Printed 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 3 2 e f 110 Bi County Blvd Ste 122 Farmingdale NY 11735 a reference then trypsinize and collect them into a 15 ml conical tube Count the cells in a hemocytometer Container Cell Number x 10 96 well plate 0 3 0 6 well 24 well plate 1 3 well 12 well plate 3 6 well 6 well plate 5 10 well 60 mm dish 20 30 100 mm dish 50 100 150 mm dish 150 180 Centrifuge the cells at 1000 rpm for 5 min Discard the supernatant Wash cells with 10 ml of PBS once by centrifugation at 1000 rom for 5 min Discard the supernatant Note For cells that are not cross linked go directly to Step 3 1 i after Step 3 1 c Add 9 ml fresh cell culture medium containing formaldehyde to 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 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 Discard the supernatant Add Working Lysis Buffer to re suspend the cell pellet 200 ul 1x10 cells and incu
21. of tissues Disaggregate tissue pieces with 20 40 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 to Step 4a 4 Cell Lysis and Chromatin Extraction Carefully remove supernatant Add CB ChIP Buffer to re suspend the chromatin pellet 100 pl 1x10 cells or 50 mg tissue 500 ul maximum for each vial Page 9 P 2027 Transfer the chromatin lysate to a 1 5 ml vial and incubate on ice for 10 min and vortex occasionally 5 Chromatin Shearing Resuspend the chromatin lysate by vortexing b Shear chromatin using one of the following methods Waterbath Sonication Epigentek EpiSonic 1100 Epigentek Cat No EQC 1100 Use 50 ul of chromatin lysate per 0 2 ml tube or per PCR plate well Shear 20 cycles under cooling condition 15 seconds On 30 seconds Off each at 170 190 watts For more detailed information of use please see the Chromatin Shearing Protocol for EpiSonic 1100 If using other waterbath sonicators please follow the supplier s instruction Probe based Sonication Use 300 ul of chromatin lysate per 1 5 ml microcentrifuge tube As an example sonication can be carried out with a microtip attached to a Branson 450 sonifier set to 25 power output Sonicate 3 4 pulses of 10 15 seconds each followed by 30 40 seconds rest
22. of unique chimeric proteins containing the maximum number of IgG binding domains coated on the strip wells This allows strong binding of any IgG subtype antibodies within a wide pH range regardless if they are in monoclonal or polyclonal form e 96 well plate format makes the assay flexible Either a manual with one single reaction each time or b high throughput with 24 48 reactions each time 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 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 e Highly efficient enrichment The enrichment ratio of positive to negative control is gt 500 An extremely low number of cells as low as 2 000 cells per ChIP reaction can be used for enriching highly abundant protein DNA complexes e High reproducibility Pre optimized ChIP conditions make the ChIP procedure consistent e Wide downstream analysis compatibility Compatible with various downstream analysis workflows including ChIP PCR ChIP on chip and ChIP seq PRINCIPLE amp PROCEDURE The ChromaFlash High Sensitivity ChIP Kit contains all necessary reagents required for carrying out a successful chromatin immunoprecipitation starting from mammalian cells or tissues This kit includes a positive control antibody RNA polymerase Il a negative control non immune IgG and GAPDH p
23. rimers 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 antibody but not by non immune IgG Immunoprecipitated DNA is then cleaned released and eluted Eluted DNA can be used for various downstream applications such as ChIP PCR ChIP on chip and ChIP seq Cell lysis and DNA shearing Protein antibody immunoprecipitation Clean protein DNA complex and reverse cross link Capture and cleaning of DNA Sequencing PCR or microarray Schematic Procedure for Using the ChromaFlash High Sensitivity ChIP Kit P 2027 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 5 Printed 2015 67 30 P 2027 805 704 E Non immune IgG 605 E ER alpha antibody 50 5 40 30 20 Relative Enrichement Fold 104 0 50 000 500 000 Cell Number Low abundance protein enricnment Sheared chromatin isolated tromaiterent numbers ot MUr cells was used for ChIP qPCR analysis of ER a enrichment in TFF1 promoters using the ChromaFlash High Sensitivity ChIP Kit Cat No 2027 and the EpiQuik Qua
24. small clearance pestle Variable temperature waterbath or incubator oven Thermocycler with 48 or 96 well block Centrifuge including desktop centrifuge up to 14 000 rpm Adjustable pipette and multiple channel pipette Aerosol resistant pipette tips 0 2 ml or 0 5 ml PCR vals oO oO oO oO oO oO oO oO oO O Orbital shaker O 1 5 ml microcentrifuge tubes O 15 mliconical tube O Cells or tissues O Antibodies of interest O Cell culture medium O 37 formaldehyde if cross linked O 1 25 M glycine solution if cross linked O 100 ethanol oO 1X PBS GENERAL PRODUCT INFORMATION Quality Control Each lot of the ChromaFlash High Sensitivty ChIP 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 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 2015 07 30 Epigentek Group Inc All rights reserved Products are for research use only P 2027 Safety Suitable lab coat disposable gloves and proper eye prot
25. x and centrifuge at 1000 rom for 5 min Remove medium and wash cells once with 10 ml of ice cold PBS by centrifuging at 1000 rpm for 5 min Discard the supernatant Add Working Lysis Buffer to re suspend the cell pellet 200 ul 1x10 cells and incubate on ice for 10 min Note If the total solution volume is less than 1 5 ml transfer the solution to a 1 5 ml microtube Vortex vigorously for 10 sec and centrifuge at 3000 rom for 5 min Then go to Step 4a 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 3 3 j after Step 3 3 b Transfer tissue pieces to a 15 ml conical tube Prepare cross link solution by adding formaldehyde to cell culture medium to 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 for every 50 mg tissues Incubate at room temperature for 15 20 min ona rocking platform Add 1 ml of 1 25 M glycine for every 9 ml of cross link solution 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 0 5 ml Working Lysis Buffer for every 50 mg
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