User Manual - Biomol GmbH
Contents
1. E02 to the dish and swirl to mix thoroughly Incubate at room temperature for 5 minutes Aspirate as buffer as much as possible NOTE From this point forward keep the samples on ice at all times e Wash the fixed cells with 10 mL ice cold 1X PBS rocking the dish for 5 seconds Pour off the wash buffer Repeat once more Aspirate as much buffer as possible Technical Support 888 503 3187 US 301 682 9200 7 ChampionChIP One Day Kit 2 Harvesting Cells a Add 0 8 mL ice cold Cell Harvesting Buffer to the dish o0 Scrape cells with a silicone cell scraper or rubber policeman Place dish on ice at an angle to scrape suspended cells down to one edge of the dish Transfer the cell suspension to a 2 mL Sonication Tube on ice Add another 0 7 mL ice cold Cell Harvesting Buffer and repeat cell scrape as above Transfer and pool the cell suspension into the same 2 mL Sonication Tube Pellet the cells by centrifuging at 800 x g for 10 min at 4 C Remove the supernatant Repeat harvesting and centrifugation steps as many times as necessary to collect all of the cells Otherwise continue with next step OR store at 80 C 3 Cell Lysis a If samples were previous frozen and stored thaw on ice Add 2 2 uL Protease Inhibitor Cocktail PIC to 420 pL IP Lysis Buffer E03 in a standard 1 5 ml Eppendorf tube Mix well by pipetting up and down Add entire volume of IP Lysis Buffer with PIC to the cell pellet 100 uL IP Ly2. c Vortex the tubes and centrifuge briefly to collect material at the bottom of their tubes d Incubate at room temp for 10 minutes e Add 50 uL of DNA Extraction Beads to each tube Vortex for about 10 s f Incubate on thermomixer shaking at 500 rpm at 95 C for 10 minutes or incubate on other incubator alternatively g Centrifuge the tubes at 14 000 x g at room temp for 1 min h Transfer 20 pL of each supernatant to a new tube i Add 4 uL 6 x DNA Loading Dye and mix well j Load samples into separate wells of the gel and proceed to gel analysis k The appropriate sonication conditions should yield sheared chromatin DNA running as a smear with an average size between 1 to 3 kb but no larger than 10 kb Technical Support 888 503 3187 US 301 682 9200 13 ChampionChIP One Day Kit Notes Technical Support www sabiosciences com support sabiosciences com 14 Version 1 2 Notes Technical Support 888 503 3187 US 301 682 9200 15 ChampionChIP One Day Kit User Manual Part 1035A Version 1 1 9 12 2008 Messer c Pal quad P P fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com supporte SABiosciences com 16
3. C Technical Support 888 503 3187 US 301 682 9200 11 ChampionChIP One Day Kit 2 DNA Purification NOTE Add 7 2 mL ethanol to the Column Wash Buffer E34 bottle and mix well before use a b C Thaw supernatants on ice if necessary Place labeled DNA Spin Columns into 2 mL Collection Tubes Add 400 uL Column Binding Buffer E33 to each 200 uL ChIP amp Input Fraction Mix well by pipetting Immediately apply each sample to its own DNA Spin Column Centrifuge at 11 000 x g for 1 min at room temp Remove DNA Spin Column from Collection Tube Discard flow through material Place DNA Spin Column back into the same Collection Tube Add 600 uL Column Wash Buffer E34 containing ethanol to each column Centrifuge at 11 000 x g for 1 min at room temp Remove DNA Spin Column from Collection Tube Discard flow through material Place DNA Spin Column back into the same Collection Tube Rotate column 180 degrees within its collection tube Centrifuge at 11 000 x g for 2 min at room temp to remove any residual volume NOTE nsure that the tip of the DNA Spin Column DOES NOT come into contact with the flow through material while removing it from the centrifuge and its Collection Tube h i j k Remove the DNA Spin Column from its Collection Tube and open the column cap Place the columns on a laboratory wipe and let it air dry 5 min Place the DNA Spin Columns into a NEW 1 7 mL Non Stick Elution Tu
4. Pre Cleared Chromatin supernatant to NEW ice cold 5 mL Pre Clear Tube To avoid disturbing the bead pellet use a P 1000 pipette tip to withdraw 900 pL three times following by P 200 pipette tip to withdraw the remaining volume Continue immediately with the next step 2 ChIP Fractions a b C d INPUT Fraction Transfer 10 uL or 1 96 of each Pre Cleared Chromatin to its own 1 7 mL Non Stick Elution Tube Store at 4 C until ready for DNA Isolation amp DNA Purification Transfer equal volume aliquots of the Pre Cleared Chromatin into three separate labeled 1 7 mL Non Stick Elution Tubes approximately 1 mL aliquot per tube Add the appropriate antibody into each ChIP Fraction i Negative Control ChIP Fraction Add 4 ug Non Immune Serum NIS ii Target Specific ChIP Fraction Add 4 ug antibody of interest e g Anti Human p53 ii Positive Control ChIP Fraction Add 4 ug anti RNA Polymerase ll antibody Incubate ChIP Fractions on rotator at 4 C for 2 hours or overnight if necessary NOTE The antibody incubation time depends on the antibody the physical number of targets and other experiment specific variables Follow the antibody manufacturer s recommendations Technical Support 888 503 3187 US 301 682 9200 9 ChampionChIP One Day Kit 3 Immunoprecipitation a To insure a homogeneous suspension invert the Protein A Beads E07 tube several times and pipette up and down 5 times using a P 200 l
5. ChIP One Day Kit I Background and Introduction Chromatin Immunoprecipitation ChIP is rapidly becoming a very important method for understanding the mechanisms of gene regulation by transcription factors and modified histones However this preparative process is very tedious and time consuming to perform involving many steps and variables that must be optimized by each investigator in their model system After crosslinking cells with formaldehyde chromatin containing covalent complexes between genomic DNA and all nuclear factors is isolated and sheared by sonication into manageable sizes Immunoprecipitation pulls down not only the target nuclear factor of interest but also any specifically bound genomic DNA sequences Reversal of the chemical crosslinking and nucleic acid purification prepare the DNA for detection by sequencing hybridization based microarrays or PCR To help researchers a number of commercial preparation kits are available that include all the reagents needed for this process While some of these kits still rely on the traditional multi day protocol to isolate ChIP DNA others offer an innovative one day solution However most kits demonstrate the success of the experiment with agarose gel based electrophoresis rather than the gold standard for nucleic acid detection real time PCR Our ChampionChIP One Day Kit is designed to help any biological researcher isolate and purify DNA bound to their nuclear factor of interest wi
6. arge bore wide mouth pipette tip immediately before each volume withdrawal Using a new P 200 large bore wide mouth pipette tip add 60 uL Protein A Beads to each ChIP fraction Incubate on a rotator at 4 C for one hour Centrifuge the ChIP fractions at 4000 x g for 1 min at 4 C Carefully place the tubes on ice for 1 min to let the Protein A Beads settle completely Remove and discard the supernatant from each ChIP Fraction To avoid disturbing the bead pellet use a P 1000 pipette tip to withdraw 900 uL following by P 200 pipette tip to withdraw the remaining volume Add 1 ml IP Wash Buffer I E21 Incubate on a rotator at room temp for 4 min Centrifuge at 4000 x g for 1 min at room temp to pellet the beads CAREFULLY remove and discard the supernatant WITHOUT disturbing the beads Add 1 ml IP Wash Buffer II E22 Repeat incubation centrifugation and supernatant removal as described for IP Wash Buffer I Add 1 ml IP Wash Buffer III E23 Repeat incubation centrifugation and supernatant removal as described for IP Wash Buffer I Add 1 ml IP Wash Buffer IV E24 Repeat incubation centrifugation and supernatant removal as described for IP Wash Buffer I Repeat IP Wash buffer IV once more and CAREFULLY remove and discard as much supernatant as possible this time Technical Support www sabiosciences com support sabiosciences com 10 Version 1 2 C DNA Isolation amp Purification This secti
7. bes Add 100 uL Elution Buffer to each DNA Spin Column Incubate at room temperature for 1 min Centrifuge at 11 000 x g for 1 min at room temp m Repeat the elution step with another 100 uL of Elution Buffer for a final 200 uL IP n DNA Fraction or Input DNA Fraction volume for each sample The purified DNA Fractions can be stored at 20 C or 4 C D ChIP DNA Detection amp Data Analysis NOTE Please refer to the ChIP qQPCR Assay User Manual for details Use 2 uL of each appropriate IP DNA fraction and Input DNA Fraction as the template for a 25 uL real time PCR assay However the amount of PCR template can range from 0 5 to 4 uL depending on the amount of input chromatin and the antibodies IP efficiency VI If you Troubleshooting amp FAQ have any questions please contact a Technical Support Representative by phone at 1 888 503 3187 or 301 682 9200 or by email at support sabiosciences net Technical Support www sabiosciences com support sabiosciences com 12 Version 1 2 Appendix A Chromatin Shearing Optimization Chromatin shearing conditions need to be optimized for your cell line of interest BEFORE performing chromatin immunoprecipitation Mock samples for optimizing shearing conditions may be prepared using untreated cells and protocol steps for Cell Cross linking Harvesting and Lysis in this manual scaling up as needed The Sonicator 3000 MISONIX Part S3000 with 1 16 microtip is rec
8. ble for any missing items not reported within two 2 business days upon receipt STORE AT 20 C CENTRIFUGE STORE AT 20 C CENTRIFUGE Tube ID Contents Quantity E01 10X PBS One 30 mL Bottle PIC Protease Inhibitor Cocktail One 1 5 mL Clear Capped Tube E02 10X Stop Buffer One 15 mL Bottle E03 IP Lysis Buffer One 15 mL Bottle E04 IP Buffer A One 15 mL Bottle E07 Protein A Beads One 2 mL Blue Capped Tube E21 IP Wash Buffer One 15 mL Bottle E22 IP Wash Buffer II One 15 mL Bottle E23 IP Wash Buffer III One 15 mL Bottle E24 IP Wash Buffer IV One 30 mL Bottle E30 ChIP Grade Proteinase K One 1 5 mL Red Capped Tube E31 DNA Extraction Beads One 2 mL Green Capped Tube E32 Elution Buffer Three 2 mL Yellow Capped Tubes E33 Column Binding Buffer One 15 mL Bottle E34 Column Wash Buffer One 15 mL Bottle DNA Spin Columns One Bag of 12 Columns Non Stick Elution Tubes 1 7 mL Three Bags of 12 Tubes 2 mL Tubes Sonication One Bag of 6 Tubes 5 mL Tubes Pre Clear One Bag of 6 Tubes Storage Conditions The ChampionChIP One Day Kit is shipped with ice packs Protease Inhibitor Cocktail PIC amp ChIP Grade Proteinase K E30 must be stored at 20 C All other components must be stored at 4 C in their original container to insure that no components are misplaced Upon arrival centrifuge Protein A Beads E07 and DNA Extraction Beads E31 at 4000 x g for 1 min a
9. fy SABiosciences E MN WWW T a User Manual ber MM Faxe 449 40 85326022 0 0800 2466652 D ChampionChIP One Day Kit Chromatin Immunoprecipitation Protocol for Quantitative Real Time PCR See Purchaser Notification for limited use license and warranty information page 3 Part 1035A Version 1 1 9 12 2008 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA ChampionChIP One Day Kit Chromatin Immunoprecipitation Protocol for Quantitative Real Time PCR User Manual For Catalog Number GA 101 aes O bi i 22769 Hamburg IOomo info biomol de Ordering and Technical Service Contact Information phone r40 408532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www sabiosciences com e E MAIL order sabiosciences net To place an order support sabiosciences net For technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www sabiosciences com SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA CONTENTS l Background and In
10. ommended for chromatin sonication in this protocol 1 Chromatin Shearing a Prepare approximately 420 uL cell lysate samples by following the protocol b Transfer 10 uL of Unsheared Control DNA to a 1 7 mL microcentrifuge tube labeled SO c Place the tube containing the remaining cell lysate sample on an ice ethanol bath rack or a rack with wet ice d Insert the probe 1 16 microtip to a 2 3 mm depth into the cell lysate e Shear chromatin with following settings Power 0 5 W Time 2 sec ON 15 sec OFF 16 sec total time or eight times per round f After the round mix the cell lysate by pipetting up and down Transfer 10 uL of the sheared chromatin to a 1 7 mL microcentrifuge tube labeled Sf g Repeat the sonication round twice saving 10 uL of the sheared chromatin each time into 1 7 mL microcentrifuge tubes labeled S2 and S3 respectively NOTE f sample starts to bubble Stop the sonication immediately Centrifuge the sample at 6000 x g for 5 min at 4 C to eliminate bubbles Continue the sonication with previous settings If bubbling continues adjust the settings to the following Power 2 5 W Time 10 sec ON 30 sec OFF Total Time 1 to 3 min 6 to 18 times per round For example use a 3 min setting and 18 times per round if foaming happens the first time 2 Agarose Gel Electrophoretic Analysis a Prepare a 1 2 agarose gel b Add 2 uL ChIP Grade Proteinase K and 8 uL ddH5O to the tubes SO S1 S2 and S3
11. on describes crosslink reversal and the isolation of DNA from the ChIP Fractions and Input DNA Fraction with sufficiently high quality and yield for real time PCR 1 DNA Isolation a Add 20 uL Elution Buffer E32 and 2 uL ChIP Grade Proteinase K E30 directly to the each ChIP Fraction and to the previously saved and stored Input Fraction Cap the tubes Vortex to mix well Incubate on Thermomixer shaking at 500 rpm or water bath or oven at 45 C for 30 min Remove the samples from the Thermomixer water bath or oven Add 100 uL DNA Extraction Beads E30 directly to each sample To insure a homogeneous suspension invert beads tube several times and pipette up and down 5 times using a P 200 large bore wide mouth pipette tip immediately before each volume withdrawal e f Cap the tubes Vortex for 10 seconds Incubate on Thermomixer shaking at 500 rpm or water bath or oven 95 C for 10 min Centrifuge samples at 14 000 x g for 1 min at room temp Transfer 90 uL of each supernatant to a NEW labeled 1 7 mL Non Stick Elution Tube without disturbing the pellet Add another 110 pL Elution Buffer E32 to each tube still containing beads Vortex for 20 seconds Centrifuge at 14 000 x g for 1 min at room temp Transfer 110 uL of each supernatant to pool the supernatants in the new tubes for a final 200 uL volume supernatant for each sample Continue with next step OR store the supernatants at 20 C or 4
12. port sabiosciences com Version 1 2 B Protein DNA Immunoprecipitation IP This section describes the immunoprecipitation of the nuclear protein s of interest with a specific antibody or antibodies This protocol requires a 100 uL aliquot of ChIP Ready Chromatin 50 ug chromatin or 2 million cell equivalents per each of three ChIP fractions one mock or negative control one positive control such as RNA Polymerase Il and one target of interest Please scale all recipes down or up accordingly to number of immunoprecipitations performed 1 Pre Clear a b C TQ T Thaw aliquots of ChIP ready chromatin on ice if necessary Add 2 7 mL IP Buffer A E04 into an ice cold 5 mL Pre Clear Tube Add 15 uL PIC and mix well by pipetting Add a 300 uL aliquot of ChIP Ready Chromatin to the 5 mL Pre Clear Tube Mix well by pipetting To insure a homogeneous suspension invert the Protein A Beads E07 tube several times and pipette up and down 5 times using a P 200 large bore wide mouth pipette tip immediately before each volume withdrawal Using a new P 200 large bore wide mouth pipette tip add 150 uL Protein A Beads to the 5 mL Pre Clear Tube or 50 uL Protein A Beads per ChIP fraction Cap the tube tightly and incubate on rotator at 4 C for 50 min Centrifuge the samples at 4000 x g for 1 min at 4 C Carefully place the tube on the ice for 1 min to let the Protein A Beads settle completely Transfer
13. sis Buffer with PIC for every 0 5 to 1 5 million cells Resuspend cells completely by pipetting up and down Incubate on ice for 10 15 min pipetting every 5 minutes Continue with next step OR store at 80 C 4 Shearing Chromatin NOTE This protocol describes recommendations for the Sonicator 3000 MISONIX Parts S3000 with a 1 16 microtip The sonication conditions need to be optimized for different sonicators and different cell lines The final cross linked sheared chromatin DNA should have an average size between 1 to 3 kb and be no larger than 10 kb as characterized on a 1 2 96 agarose gel Please see Appendix A for details on optimizing this step a b C Thaw cell lysate on ice if necessary Place the sample tube in a wet ice ethanol bath or wet ice bath Insert the probe 1 16 microtip to a 2 3 mm depth into the cell lysate Shear cell lysate using optimized conditions For example cell lysate can be sheared under the following settings Power 0 5 W Time 2 sec ON 15 sec OFF Total Time 16 sec 8 times per round 3 rounds Mix the cell lysate by pipetting up and down after every round d e f Pellet the debris by centrifugation at 14 000 x g for 10 min at 4 C CAREFULLY transfer the supernatant to a new 1 7 mL Non Stick Elution Tube without disturbing the debris pellet Continue with next step OR snap freeze the ChIP Ready Chromatin at 80 C Technical Support www sabiosciences com sup
14. t 4 C and return to their original position in the box Shelf Life All reagents are stable for 6 months after receipt of the kit if stored at the recommended temperature Technical Support 888 503 3187 US 5 301 682 9200 ChampionChIP One Day Kit Ill Additional Materials amp Equipment Required Additional Materials Required The following components are needed for the protocol but are not included in the kit e 37 Formaldehyde Sigma Catalog Number F1635 e Silicone Cell Scraper or Rubber Policeman VWR e Filter Tips e P 200 large bore wide mouth pipette tip VWR Cat 46620 642 in North America or 732 0544 in Europe e Wet ice ethanol bath or wet ice bath Equipment Required e Sonicator Rotator Thermomixer Eppendorf or shaking water bath UV Spectrophotometer Centrifuge Bench top Microcentrifuge Rotating Platform at 4 C and room temp Real Time PCR Instrument Technical Support www sabiosciences com support sabiosciences com 6 Version 1 2 IV Protocol Notes on Sample Preparation The protocol for sample preparation depends on the nature or source of the sample itself and the experiment design Generally each ChIP Assay needs at least two ChIP fractions one mock or negative control and one for the target of interest and sometimes an optional third fraction as a positive control for example RNA Polymerase II Each ChIP fraction requires 1 2 million cells This protocol is based on
15. th the quality required by real time PCR detection The complete preparation kit includes all buffers and components needed for immunoprecipitation crosslink reversal and genomic DNA purification for detection The general universal kit also offers compatibility with any chosen ChIP grade antibody Otherwise all you need is cross linking agent a sonicator and your cell based experiment The protocol and reagents have been carefully optimized to take most of the guesswork out of ChIP preparation Tips and hints are also provided in the protocol for optimizing your cell harvesting and sonication conditions steps that you must control and the kit cannot The entire protocol can be completed in one day but also includes convenient stopping points for continuing the protocol the next day The resulting DNA is free of contaminants that confound and interfere with real time PCR detection in particular using the ChampionChIP Real Time PCR Primers Benefits of the ChampionChIP One Day Kit e Speed amp Ease Simplified protocol easily obtains ChIP DNA from cells in just 6 hours e Universality General kit compatible with any ChIP Grade antibody e Reliability High yield and high quality DNA for real time PCR and highly reproducible enrichment Technical Support www sabiosciences com support sabiosciences com 4 Il Materials Provided Version 1 2 Please check the kit components immediately after you receive this package We are not responsi
16. the use of 4 6 million adherent mammalian cells from one experimental sample enough for 3 to 4 ChIP fractions Please scale all recipes down or up according to the number of cells that you are harvesting A ChIP Ready Chromatin Preparation This section describes the preparation of ChIP Ready Chromatin It covers cell cross linking harvesting lysis and chromatin shearing based on adherent mammalian cell lines Certain steps may need to be optimized for experiment dependent performance This protocol also provides several stopping points for the flexibility to optimize those steps 1 Cross Linking Cells a Grow cells under normal conditions to 70 85 confluence For example HeLa cells at 70 85 confluence on a 10 cm dish number roughly 4 to 6 million cells Prepare one extra plate of cells to estimate cell number if necessary b Before harvesting cells prepare following buffers scale up accordingly if necessary i 1X PBS Buffer Add 3 3 mL 10X PBS E01 to 29 7 mL ddH2O Mix well and place on ice ii FRESH Fixing Buffer 1 Formaldehyde Add 0 27 mL 37 Formaldehyde to 9 73 mL 1 x PBS Buffer Mix and store at room temperature in a fume hood ii Cell Harvesting Buffer Add 15 uL Protease Inhibitor Cocktail PIC to 3 mL ice cold 1 x PBS Buffer Mix well and place on ice c Aspirate cell culture medium from the dish Add 10 mL FRESH Fixing Buffer to the dish Incubate at 37 C for 10 minutes d Add 1 1 mL Stop Buffer
17. troduction 4 I Materials Provided 6 Il Additional Materials Required T V Protocol 8 A ChIP Ready Chromatin Preparation 8 B Protein DNA Immunoprecipitation IP 10 C DNA Isolation amp Purification 12 D ChIP DNA Detection amp Data Analysis 13 VI Troubleshooting amp FAQ 14 Appendix Chromatin Shearing Optimization 15 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of ChampionChIP One Day Kit includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of the kit to modify kit components for resale or to use the ChampionChIP One Day Kit to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppels is granted Champion
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