ChIP doesn't work. Please help! - Too much "normal rabbit IgG" bockround (Feb/08/2011 )

hi @ all,

I'm new to this forum and desperatly looking for help because I've now tried to establish ChIP in our lab for almost more than 4 months and I'm not getting it.
After trying MNase digestion I got back to Sonification and found good settings (with Covaris S2; see below for details).
I tried the immunoprecipitation with magnetic beads and also with Agarose beads. In both cases my "normal rabbit IgG"-control is higher than my precipitations. So theres no enrichment due to the IP but instead an decrease...
I use same amounts (2 µg) of antibody. I asked different people and got another (unpublished) protocol where they use only 5% volume of Chromatin for the IgG isotype control (and 1% volume for Input, 95% for IP of interest). is this ok? I don't think I'm allowed to handle the problem this way?

So my questions:

- Why do I have such an huge "normal rabbit IgG"-control backround? (Much more higher than the IP of interest

- Is it ok to use only 5% of chromatin for the IgG isotype control?

I would be very very happy if somebody could help me....





my (own) protocol looks like this:

A.) Crosslinking of Cells

- Transfer ~5 X 107 cells to 50 ml conical tube in 25ml media
- On Belly Dancer, add 0.67ml 37% formaldehyde dropwise to cells (1% final concentration), let incubate 10min at RT
- Add 1.25ml 2.5M glycine dropwise to shaking cells (1X final concentration), continue to shake 5’
- Centrifuge 10 min @ 1100 rpm and 4°C
- Wash pellet in 10 ml
- Discard supernatant and wash Pellet in 10 ml cold PBS with 5 µl PIC
- Centrifuge 10 min @ 1100 rpm and 4°C
- Discard supernatant and wash Pellet in 10 ml cold PBS with 5 µl PIC
- Centrifuge 10 min @ 1100 rpm and 4°C
- Discard supernatant and add 5 µl PIC and 5 µl PMSF
- Store Pellet @ -70°C

B.) Chromatin preparation

- Resuspend frozen pellet in 1 ml ice-cold Lysis Buffer + 5l each of 100mM PMSF and 100X PIC, incubate on ice 30’
- transfer to 1.7 ml microfuge tube, pellet nuclei 10’ @ 5K RPM @ 4oC
- Remove supernatant, resuspend nuclei in 0.5 ml MNase Buffer + 2.5l each of 100mM PMSF and 100X PIC
- Add 50 µl of 1% SDS Solution (final conc. Of 0.1%)
- Measure OD260 (NanoDrop) of 1:10 dilution
- pellet nuclei 10min @ 600 x g @ 4oC
- Resuspend pellet in MNase Buffer + 1:200 each of 100mM PMSF and 100X PIC to final concentration of 0.5mg/ml, incubate 5’ @ 37oC

C.) Sonification

- Store between sonification on ice
- Sonification with „Covaris S2“


Number of Cycles Duty Cycle Intensity Cycles per Burst Time
30 20 9 500 60

D.) Control of Sonification

- To check shearing, add 150 l H20 to 50 l chromatin. Add 8 l 5M NaCl, 3 l RNAse A (10 mg/ml). Vortex and incubate 30 min @ 37°C.
- Add 2.5 l (20 mg/ml) Proteinase K, incubate 2h @ 62°C
- Purify DNA with the “Quiagen PCR Purification Kit

E.) Preperation of the magnetic beads

- After resuspension (the Prot. A beads stick to the glass, so be thorough) give 20l Protein A magnetic beads per IP in 1.5ml micro-centrifuge tube, place on magnet aspirate media
 for 4 IPs = 80 µl
- Wash beads 2X in 100l 1X RIPA/ssDNA per IP (each time mix by moving back and forth across magnet several times then place on magnet and aspirate)
 for 4 IPs = 400 µl
- Resuspend bead in 50l 1X RIPA/ssDNA per IP
- Add 0.5l PMSF (100mM in EtOH) and 0.5l 100X PIC per IP
- Add 1-5 g (depending on antibody) of specific antibody or IgG per IP (here 2 µg used)
1. Egr3 (200 µg/ml)  10 µl
2. Myc (290 µg/ml)  6,89 µl
3. IgG (1 mg/ml)  2 µl
4. RNA-Pol (1,34 mg/ml)  1,49 µl
- Rotate 1hr to oN at 4C

F.) Immunoprecipitation

- Wash Ab:bead conjugates 3X in 100l 1X RIPA/ssDNA per IP tube
- Resuspend in 90l 1X RIPA/ssDNA per IP. Add 1l PMSF and 1ul 100X PIC per IP
- Add 10l chromatin to each tube rotate 2hr at 4
- Place on magnet and aspirate media
- Wash chromatin:Ab:Beads 3-4X in 180l 1X RIPA (no ssDNA)
- Wash chromatin:Ab:Beads 2X in 180l TE pH8.0 (Don’t go slow here or you will lose beads on the second wash—they don’t bind the magnet well in TE)
- Resuspend Chromatin:AB;Beads in 100l fresh 100mM NaHCO3/1%SDS and vortex lightly for 15 minutes to elute chromatin:antibody from beads
- Add 2l 5M NaCl and immediately place on magnet and transfer media to fresh tubes. (Actually, here I usually put the salt in the new tube, place the elution tubes on the magnet and transfer the supernantant to the salt-containing fresh tube). Incubate 15’ at 95C to reverse formaldehyde crosslinks (the eluates could be frozen at -20oC once the magnetic beads are removed—either before or after the 15min at 95oC step)
- Cool down the samples
- Add 1l Proteinase K (20 mg/ml) and incubate 1hr at 42C, stop in 2l PMSF (at this point, the DNA is stable and can be stored at -20oC)
- Purify on Qiagen PCR Purification kit and resuspend in 100l H20 (eluate two times with 50 µl; at this point, the DNA is stable and can be stored at -20oC).

G.) qRT-PCR

- Use the same volume for qRT-PCR
- Also perform for Input


Solutions:

Lyse Buffer MNase Buffer
100 µl 1 M Tris (pH=7,5) 500 µl 1 M Tris (pH=7,5)
30 µl 1 M MgCl2 40 µl 1 M MgCl2
20 µl 5 M NaCl 10 µl 1 M CaCl2
500 µl 10% NP40 3,33 ml 1 M Sucrose
9,35 ml H2O 50 µl 1 M Na Butyrate
6,1 ml H2O


37% Formaldehyd 10x RIPA
1,85 g Paraformaldehyd 100 mM Tris-HCl, pH=7,5
70 µl 1 M KOH 1,5 M NaCl
4,8 ml H2O 10 mM EDTA
10% Triton X-100
1% SDS
1% NaDeoxycholate

1x RIPA/ssDNA
1 ml 10x RIPA
1ml 5mg/ml salmon sperm DNA
8 ml PCR grade water




F’.) IP

1.) Split the remaining chromatin solution (450 μl) into 3 tubes with 150 μl each. Add 1,350 μl ChIP dilution buffer to each tube (note: to dilute SDS from 1% to 0.1% in the buffer).
2.) For each tube from step 29, prepare a 40 μl of 50% salmon sperm DNA/ protein A agarose beads by rinsing three times with 1 ml of ChIP dilution buffer (without protease inhibitor or sodium butyrate) and resuspend the beads in 40 μl of ChIP dilution buffer (note: after each wash, spin down beads at 6,000 rpm for 2 minutes at 4°C).
3.) Incubate the chromatin solution (1.5 ml) with 40 μl of protein A agarose beads for 1 hour at 4°C in a rotisserie shaker.
4.) Centrifuge at 6,000 rpm for 2 minutes at 4°C to pellet beads from the chromatin solution.
5.) Combine 3 aliquots of chromatin solution into a 14 ml falcon tube (note: avoid pipetting pellet protein A agarose beads).
6.) For each chromatin IP, mix 600 μl pre-cleared chromatin solution with antibody in a 1.5 ml microcentrifuge tube (notes: the amount of antibodies used varies among different sources and should be determined empirically; we used 10 μl anti-H3K9Ac, 5 μl anti-H3K9Me2, 5 μg anti-H3K4Me2 or 10 μl anti-CCA1 per IP).
7.) Set up another chromatin solution (600 μl) without antibodies as a control for ChIP.
8.) Incubate chromatin and antibodies overnight at 4°C in a rotisserie shaker.
9.) Prepare 40 μl of 50% salmon sperm DNA/protein A agarose beads as in step 30.
10.) Add 40 μl of protein A agarose beads to the ChIP tube and incubate for 1.5 hours at 4°C in a rotisserie shaker (note: pull down the ChIP-complexes formed).
11.) Centrifuge at 6,000 rpm for 1 minute at 4°C to collect ChIP-beads complexes, discard supernatant.
12.) Add 1 ml of low salt buffer to ChIP-beads complexes.
13.) Wash the complexes for 10 minutes at 4°C with rotation.
14.) Centrifuge at 6,000 rpm for 1 minute at 4°C to collect ChIP-beads complexes, discard supernatant
15.) Add 1 ml of high salt buffer to ChIP-beads complexes
16.) Repeat steps 13 and 14
17.) Add 1 ml of LiCl washing buffer to ChIP-beads complexes
18.) Repeat steps 13 and 14

19.) Add 1 ml of TE buffer to IP-beads complexes
20.) Repeat steps 13 and 14
21.) Repeat washing with TE buffer (steps 19 and 20), collect ChIP-beads complexes and discard supernatant.
22.) Add 250 μl of elution buffer to elute ChIP complexes (note: prepare elution buffer prior washing steps and store it at 65°C).
23.) Vortex briefly and incubate the samples at 65°C for 15 minutes.
24.) Centrifuge the samples at 13,000 rpm for 2 minutes at room temperature.
25.) Carefully transfer the supernatant (eluate) to a new 1.5 ml screw cap tube.
26.) Repeat elution (steps 50 to 53) and combine the two eluates in a single tube.
27.) Add 20 μl of 5 M NaCl to the eluate and reverse cross-link at 65°C for at least 6 hours to overnight (note: also perform reverse-crosslink for input DNA control from step X).
28.) Add 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl (pH6.5) and 1 μl of 20 mg/ml proteinase K.
29.) Incubate at 45°C for 1-3 hours.
30.) Purify the DNA with the “Quiagen PCR Purification Kit”.

For your control I would try using another source of IgG or, even better, an antibody to an epitope that doesn't exist in the cells you're using (like an anti-Flag antibody if you're not using Flag-tagged proteins). It's possible that, by chance, your current IgG has an antibody clone which is binding a lot of chromatin.

Another thing to try is decreasing the amount of chromatin you are using, especially if you are getting really low Cts. You could try using two-fold, four-fold, and eight-fold less and see if the ratio of specific IP to control IP improves.

Finally, when you do decrease your background to acceptable levels, keep in mind that enrichment isn't determined by the ratio of specific IP to control IP or by the percent of input, but instead, by the amount of signal at your region of interest over the signal at a negative control region where your factor is not expected to bind.

Oh, and use the same amount of chromatin in your control IP as you would in your specific IP. There's no point to running the control IP if you don't use the same amount of chromatin.

whats the fold enrichment of your 1% Input over IgG? I will second KPDE's suggestion on picking an antibody with a known epitope.......especially of your looking at chromatin on non-host DNA, like viral DNA.

I have same problem!!!
I am using a rabbit-anti-Hif1a antibody, and I always get the pull down in IgG.
I increased the washing, decreased the cell number, but still doesn't work.
Is it possible that my DNA fragment bind to the IgG, too?

benrabbit on Wed Apr 6 20:57:40 2011 said:

Have you seen what your pulldown is for beads alone? If this is much lower than with IgG then it suggests there is one or more clones in your total IgG that is binding to the chromatin. In that case I would try a different sera, like a different source of IgG or an anti-flag antibody (or antibody to some other epitope that your cells don't express).

I would second the call by KPDE for the use of an anti-FLAG or similar antibody instead of IgG. I have started to use anti-myc tag as my negative control more and more as I got so sick of the IgG. It is a terrible control when you think about it.

Agreed with KDPE as well...point of emphasis from his initial response is that to determine whether your actually getting binding to your site or not you have to compare your target site to a genomic site in which you expect your factor NOT to bind (I usually normalize both to input for comparison). This is the real test as to whether or not you have TF binding...screw the IgG "control" it's a bunch of crap for determining whether your factor is binding or not.

Hi Grenouille,

I realize that this message might be too late, but I do know why your ChIP was not working:
1. you are fixing for too long, adding the concentrated formaldehyde directly to your cells, and using most likely methanol containing 37% formaldehyde which will increase the fixation rate.
2. you are shearing the chromatin at too high of an energy, so you have most likley destroyed all the epitopes. We do notice an increase in mock IP when chromatin is over processed.

thank you

Hamid

Hi,

I am trying 1% formaldehyde (same as above) for corsslinking plant tissue which are crushed in liquid nitrogen and stored at -80C. Yet, havent got any luck! What concentration of 37% formaldehyde you would suggest to use?

Thanks