《免疫共沉淀ChIP.doc》由会员分享，可在线阅读，更多相关《免疫共沉淀ChIP.doc（3页珍藏版）》请在金锄头文库上搜索。
1、ChIP protocolBasic on EZ-ChIP Chromatin Immunoprecipitation KitUpstate Catalog # 17-371A. In Vivo Crosslinking and Lysisl 1、 Stimulate or treat 4.5 x 107 cells on a 10cm dish as appropriate. (25K、5K for 4h)l 2、Cross link protein to DNA by adding formaldehyde directly to culture medium to a final con
2、centration of 1% and incubate 10 minutes at room temperature. (add 270ul 37% formaldehyde into 10ml of growth medium on plate) l 3. Add 1 ml of 10X glycine to each dish to quench unreacted formaldehyde. Swirl to mix and incubate at room temperature for 5 minutes.l 4、Aspirate medium, wash cells twice
3、 using 10ml ice cold PBS.l 5、Add 1ml cold PBS containing Protease Inhibitor to dish.l 6、Scrape cells into conical tube. Spin at 700 rcf at 4 for 2-5 minutes to pellet cells.l 7、Remove supernatant. l 8、Resuspend each cell pellet in 600ul of SDS Lysis Buffer(1%SDS, 10mM EDTA, 50mM Tris, pH8.1) contain
4、ing Protease Inhibitor. Incubate on ice for 10 minutes. (Remove 5ul of cell lysate for unsheared DNA control).(Lysate can be frozen at -80 at this step.)B. Sonication to Shear DNAl 1、Sonicate chromatin to an average length of about 500 bp while keeping samples on ice.(60% output,10s/6 times recall#2
5、0 program)l 2、Microfuge at 13,000 rpm for 10 minutes at 4.Carefully remove the supernatant and transfer to a new tube in 100ul aliquots.(Sheared crosslinked chromatin can be stored at -80C for up to 2 months.)l 3、To all the samples (unsheared and sheared), remove 5ul to a fresh tube. Add ChIP elutio
6、n buffer (1% SDS, 0.1 M NaHCO3) to a final volume of 30 ul.l 4、Add 1ul of RNase A (10 mg/ml) and incubate for 30 minutes at 37.l 5、Add 1ul Proteinase K(10 mg/ml) and incubate at 62 for 2 hour.l 6、Load 15ul on a 2% agarose gel. Observe which of the shearing conditions gives a smear of DNA in the rang
7、e of 200 bp-1000 bp.C. Immunoprecipitation (IP) of Crosslinked Protein/DNAl 1、Add 900ul of Dilution Buffer(0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, containing Protease Inhibitor) into each tube containing 100ul of chromatin.l 2、Preclear: add 30ul of Protein G
8、/A Agarose for each IP incubate 1h at 4 with rotation .(After preclear remove 50ul supernatant for input DNA) Pre-incubate: add 30ul of Protein G/A Agarose with 2ug immunoprecipitating antibody incubate 1h at 4 with rotation.(c-jun H79,ATF2 SC187x).l 3、Supernatant of preclear tube incubate with the
9、pre-incubate beads at 4 overnight with rotationl 4、Pellet Protein G/A Agarose by brief centrifugation (5000 rpm for 2 minute) and remove the supernatant fraction.l 5、Wash in 1ml each of the cold buffers and incubating for 3-5 minutes on a rotating platform followed by brief centrifugation (5000 rpm
10、for 2 minute) and careful removal of the supernatant fraction.a. Low Salt Immune Complex Wash Buffer (Catalog # 20-154), one washb. High Salt Immune Complex Wash Buffer (Catalog # 20-155), one washc. LiCl Immune Complex Wash Buffer (Catalog # 20-156), one washd. TE Buffer (Catalog # 20-157), two was
11、hesOR wash pellets five times in Wash Buffer( 20 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% TritionX-100, containing Protease Inhibitor).D. Elution of Protein/DNA Complexesl 1、Add 100ul of Elution Buffer(1% SDS, 0.1 M NaHCO3) to each tube containing the antibody/agarose complex. Mix by flicking
12、tube gently. Incubate at room temperature for 15 minutesl 2、Pellet agarose by brief centrifugation (5000 rpm for 2 minute) and collect supernatant into new microfuge tubes.l 3、Repeat again and combine eluates (total volume = 200ul). l 4、For Input tubes, add 200ul of Elution Buffer. E. Reverse Crossl
13、inks of Protein/DNA Complexes to Free DNAl 1、To all tubes (IPs and Inputs) add 8ul 5 M NaCl and incubate at 65 overnight to reverse the DNA-Protein crosslinks.l 2、To all tubes, add 1ul of RNase A and incubate for 30 minutes at 37.l 3、Add 4ul 0.5M EDTA, 8ul 1M Tris-HCl and 1ul Proteinase K to each tu
14、be and incubate at 45 for 1-2 hours.F. DNA Purification Using Spin Columnsl Use Qiagen gel extraction kit.l 1、Add 3 volume of buffer QG to 1 volume of sample, mix gently.l 2、Place a QIAquick spin column in a provided 2ml collection tube.l 3、To bind DNA, apply the sample to the QIAquick spin column,
15、and centrifuge for 1min, 7000rpm.l 4、Discard flow-through and place QIAquick spin column back to the same tube.l 5、To wash, add 750ul buffer PE to QIAquick spin column, and centrifuge for 1min, 7000rpm.l 6、Discard flow-through, and centrifuge for 1min, 13000rpm.l 7、Place QIAquick spin column into a
16、clean 1.5ml EP tube.l 8、Add 50ul buffer EB, stand for 2min, and centrifuge for 1min,13000rpm. And chromatin can be stored at -20CG. PCRl c-jun: ChIP-173-241-F, ChIP-173-Rl dp5: ChIP-F-169, ChIP-R-169l atf3:ChIP-F2-186, ChIP-R1-411c-Jun：forward 5-CTAGACAGCCAAACCAAGAC-3；reverse 5-GCTCACGGGATGAGGTAAT-3。dp5： forward 5-AGGGTTAAAAGTTACCTCTCGGC-3；
