Day 5
1.Thaw extracts on ice and transfer a volume of extract (1mg total protein)to a new 1.5 ml tube for IP
2.Include necessary controls (i.e.no Ab control; untagged strain+ Ab)
3.Bring all 1mg aliquots to 200μl with ChIP lysis buffer+protease inhibitors,mix well (thorough mixing is very important)
4.Transfer 2μl from each 200μl IP sample to a new 1.5 ml tube with 150μl ChIP elution buffer and set aside until the IP’s are complete (these are the INPUT samples)
5.Add Ab to each IP sample except for the no-Ab control,nutate in cold room for 3 hrs
6.When there is 30 min left from step 5 prepare rProtein A (or G)Sepharose beads
Spin down beads and remove supernatant
Wash beads twice with 1ml ChIP lysis buffer+protease inhibitors per 250μl bead volume
Resuspend beads after washing in an equal volume of ChIP lysis buffer+protease inhibitors to make 50% slurry (prepare 25% more beads than you calculate to be necessary)
7.Add 60μl of 50% bead slurry to each IP sample,nutate in cold room 1hr
8.Spin beads down in cold room microcentrifuge for 10-15 sec,remove the supernatant,and wash with the following solutions
2x 1ml ChIP lysis buffer+protease inhibitors
2x 1ml ChIP high salt lysis buffer+protease inhibitors
2x 1ml ChIP wash buffer
2x 1ml 1xTE (8.0)
9.After the last wash draw off as much liquid as possible from the beads using a finely pulled Pasteur pipette
10.Resuspend the beads in 85μl ChIP elution buffer and incubate at 65 degrees C and 950 rpm in a Thermomixer for 10 min
11.Spin down beads in microcentrifuge and transfer 75μl of the supernatant to a new 1.5ml tube
12.Add 75μl to the beads,repeat steps 10 and 11,and combine the two 75μl elutions (IP)
13.For each extract being tested you should now have an IP sample (150μl)and an IN sample (150μl)plus any controls
14.Incubate all IP and IN samples at 65 degrees C overnight to reverse crosslinks
Day 6
1.Add 750μl Qiagen buffer PB to each IP and IN sample
2.Purify using the Qiagen PCR purification kit,elute DNA in 50μl buffer EB
3.Analyze the samples by quantitative PCR using an appropriate protocol
--------------------------------------------------------------------------------
ChIP Buffers
2.5 M glycine (filter sterilize,store at room temperature)
1x TBS+ 125 mM glycine (store at 4 degrees C)
1x TBS (store at 4 degrees C)
ChIP lysis buffer (filter sterilize,store at 4 degrees C)
50 mM HEPES pH 7.5
140 mM NaCl
1% TritonX-100