Chromatin Immunoprecipitation (ChIP) Protocol using Protein A beads
- junji.lin@neuro.utah.edu
Chromatin Immunoprecipitation (ChIP) Protocol using Protein A beads (Dorsky Lab)
Shan-Fu’s adaptation from Kazuyuki’s protocol (Grunwald Lab) (8/24/05)
Modified by Junji Lin (06/29/2010)
<Prior to Day1>
¨ Coating protein A beads
Take 50% protein A beads slurry (about 50ul for each sample) (Invitrogen #15918-014)
Spin 1.5k rpm, 30 sec at RT
Remove the sup. with loading tip
Wash three times with 1 mL IP dilution buffer
Resuspend beads in 1mL pre-blocking buffer (10 mg/mL calf thymus DNA 100 uL, 20 mg/mL BSA 50 uL, and IP dilution buffer 850 uL) and incubate at 4oC, O/N
Spin 1.5k rpm, 30 sec at RT
Remove the sup.
Wash twice with 1 mL IP dilution buffer
Add IP dilution buffer back to original volume
(Comments: This protocol is not for ChIP-Seq since other types of carrier DNA (such as calf thymus DNA) were used for blocking. Washing and blocking protein A beads are necessary for eliminating non-specific beads’ binding.)
<Day 1>
¨ Cross-linking of protein and DNA
Dechorinate 100 embryos in 1xER, then remove the supernatant (sup.)
Add 1% formaldehyde in 1xPBS in 1.5 mL tube
Rotate 15 min at RT, remove sup.
Rinse with 1 mL 0.125 M glycine
Add 0.125 M glycine to soak embryos, rotate 10 min at RT
Rinse embryos with 1 mL cold 1xPBS twice
(Comments: The amount of embryos needed for ChIP need to be optimized by specific sample. It also depends on your antibody specificity. As far as I know, I can see difference between negative control and sample with only 10 embryos using dynabeads. But I haven’t check it with protein A beads.
Cross-linking condition are based on100 embryos at 36hpf. Time will vary if using younger or older embryos; 2.2% of formaldehyde can be used if necessary. Insufficient cross-linking will lose true binding signals and over cross-linking will bring non-specific binding. )
¨ Cell lysis and chromatin DNA extraction
Add 600 uL Cell lysis buffer (for 100 embryos), on ice 10~20 min
Pipet up and down every 5-10min until no clear tissue can be visible (bone structure might be still there, but most of the tissue should be broken and dissolved)
Cfg: 3.5k rpm, 5 min at 4 oC, remove the sup.
Rinse once more with 600 uL Cell lysis buffer and spin down, remove the sup.
Re-suspend the nuclear pellet (white) in 200 uL nuclei lysis buffer
Pipet up and down to disrupt clumps
On ice 10~20 min (lay the tube on ice to avoid precipitation of SDS in the nuclei lysis buffer)
Add 400 uL IP dilution buffer + proteinase inhibitors
Freeze at -70 or -80 oC
¨ Sonication
Thaw the sample and add 100 mg glass beads (Sigma, acid washed, 212-300 microns, Sigma #G-1277)
Sonication machine (Branson Sonifer 250 with microtip)
Set up: constant duty cycle, output on 2, value 20
6 pulses of 20 sec each with 3 min interval on ice
Take 10 uL for agarose gel electrophoresis to check the size of fragmented DNA after decross-linking
(Comments: Over sonication will lead to DNA degradation, Insufficient sonication will lead to non-specific binding. Average fragment size should be between 100bp-600bp. Avoid introducing bubbles to the sample or generating too much heat since it will lead to protein degradation. Keep on ice during sonication and leave the sample on ice for enough time to cool down before next round of sonication.)
Cfg: 14k rpm, 15 min at 4oC
Take the sup. Add IP dilution buffer to 600 uL per tube.
Divide equal proportionally for ChIP and total input control. (For example: total amount after sonication is 600ul, using 500ul for ChIP assay and 100ul for input control for each sample) Store input sample at -20 oC.
¨ Antibody binding
Pre-clear:
Add 40 uL coated protein A beads (in pre-Day1) to the ChIP sample
Rotate at 4oC, 15 min
Antibody binding:
Cfg: 5k rpm, 10 min, 4oC
Take the sup. (Do not contaminate with any beads) and divide it equally as antibody and no-antibody control.
Add antibody 5ug to the sample (Keep the no-antibody control sample at 4 oC without adding any antibody) rotate O/N at 4oC
<Day 2>
¨ Protein A beads binding, washing, elution, and decross-linking
Add 40 uL blocked 50% protein A beads slurry (also to no-antibody control tube)
Incubate on a rotating wheel/platform at RT, >15 min
Cfg: 1.5k rpm, 4 min at RT,
Remove sup. (Beads from no-antibody tube could serve as “no Ab” negative control after washing and elution)
Wash the beads twice with 1 mL 1x dialysis buffer
Add 1 mL buffer
Rotate 15 min at RT
Cfg: 1.5k rpm, 4 min at RT
Remove the sup. as much as possible with loading tip
Wash the beads twice with 1 mL IP wash buffer (same as above)
(Comment: Washing step could be held at 4 oC with longer time; If non-specific binding still occur, wash 3 times for each buffer followed by 1~3 times TE wash)
¨ Elution and decross-linking
Add 150 uL elution buffer to the beads
Rotate 15 min at RT
Cfg: 14k rpm, 4 min at RT
Transfer the sup. to a new tube
Repeat elution steps again and combine both elution (300 uL).
Add 30 uL 3 M NaCl and 1 uL 10 mg/mL RNaseA (For input sample, add right amount of NaCl and RNaseA)
Incubate 65 oC, 4-5 hr or O/N (input control samples also need to be decross-linking at the time)
Add 2 uL 5 mg/mL glycogen and 2.5V absolute EtOH, -80oC O/N
<Day 3>
¨ Proteinase K treatment
Cfg: 14k rpm, 4oC, 20 min
Discard sup.
Dissolve pellet in TE, and mix with 5x PK buffer PK(10 mg/mL)
For ChIP sample 100 uL 25 uL 2 uL
For Input sample 200 uL 50 uL 2 uL
45oC, 1-2 hr
Add TE to 300 uL
Add 300 uL phenol/CHCl3, vortex, 14k rpm, 5 min at RT, take the sup.
Add 300 uL CHCl3, vortex, 14k rpm, 5 min at RT, take the sup.
Add 3 M NaCl 54 uL, 5 mg/mL glycogen 2 uL, 2.5V absolute EtOH
-80oC, O/N
<Day4>
¨ Precipitation and PCR
14k rpm, 20 min at 4oC
Dissolve pellet in 10-30 uL TE or ddH2O
Further DNA purification can use QIAquick PCR purification Kit if necessary. But it will also lose quite a bit amount of DNA.
DNA can be stored at -20 oC
qPCR with ChIP sample, no-Ab control and total input.
¨ Recipes for solution and material information
1 M glycine (FW = 75.07)
3.75 g / 50 mL dH2O
filtrate and store at RT
100x ER
6 g instant ocean / 1 L dH2O
1% formaldehyde in 1x PBS (freshly prepared)
Total 25ml
37% formaldehyde 676 uL
1x PBS 24.324ul
0.125 M glycine in 1x PBS (freshly prepared)
10 mL 30 mL
1 M glycine 1.25 mL 3.75 mL
1x PBS 8.75 mL 26.25 mL
Proteinase inhibitors
(final conc.) (per 1 mL solution/buffer)
100 uM PMSF 1 uL of 100 mM
100 ug/mL benzamideine 10 uL of 10 mg/mL
5 ug/mL leupeptin/pepstatin 1 uL of 5 mg/mL
Cell lysis buffer (at 4oC) (40 mL)
10 mM Tris-Cl (pH 8,1) 400 uL of 1 M (Using Tris Base)
10 mM NaCl 133 uL of 3 M
0.5% NP-40 2 mL of 10%
proteinase inhibitors (freshly add)
dH2O 37.47 mL
Nuclei lysis buffer (20 mL)
50 mM Tris-Cl (pH 8.1) 1 mL of 1 M
10 mM EDTA 400 uL of 0.5 M
1% SDS (Sodium Dodecyl Sulfate) 2 mL of 10%
proteinase inhibitors (freshly add)
dH2O 16.6 mL
IP dilution buffer (at 4oC) (40 mL)
16.7 mM Tris-Cl (pH 8.1) 668 uL of 1 M
167 mM NaCl 2227 uL of 3 M
1.2 mM EDTA 96 uL of 0.5 M
1.1% Triton X-100 2.2 mL of 20%
0.01% SDS 40 uL of 10%
proteinase inhibitors (freshly add)
dH2O 34.77 mL
Pre-Blocking Buffer: 1 mg/mL calf thymus DNA and 1 mg/mL BSA in IP dilution buffer (1 mL)
10 mg/mL BSA 100 uL
10 mg/mL denatured calf thymus DNA 100 uL
IP dilution buffer 800 uL
500 mM NaHCO3 (filtrate, store at RT)
NaHCO3 (FW = 84.01) 4.2 g
H2O up to 100 mL
1x dialysis buffer (store at 4oC) (100 mL)
50 mM Tris-Cl (pH 8.1) 5 mL of 1 M
2 mM EDTA 400 uL of 0.5 M
0.2% sarkosyl (omit for monoclonal Ab) 1 mL of 20%
The other name is N-lauroylsarcosin solution (SIGMA L7414-10ML)
dH2O 92.6 mL
IP wash buffer (stored at 4oC) (100 mL)
100 mM Tris-Cl (pH 9.0) (pH 8.0 for monoclonal Ab) 10 mL of 1 M
500 mM LiCl 10 mL of 5 M
1% NP-40 10 mL of 10%
1% deoxycholic acid (Sodium deoxycholate Sigma D6750-25G) 5 mL of 20%
dH2O 65 mL
Elution buffer (40 mL)
50 mM NaHCO3 4 mL of 500 mM
1% SDS 4 mL of 10%
dH2O 32 mL of dH2O
5x PK buffer
50 mM Tris-Cl (pH 7.5) 5 mL of 1 M
25 mM EDTA 5 mL of 0.5 M
1.25% SDS 12.5 mL of 10%
dH2O 77.5 mL
Glass beads, acid-washed, Sigma G1277-100G
10 mg/mL proteinase K
10 mg/mL glycogen
3 M NaCl
Phenol (saturated with TE)
CHCl3 (+1/25 vol. Iso-amyl alcohol)
EtOH (-20oC)