Chip Troubles - Help: Strong PCR signal from negative control region (Sep/01/2008 )

Hello. I'm hoping someone might help me troubleshoot my ChIP experiment.

I'm trying to perform the following ChIP protocol for a transcription factor using a cell line that over-expresses a flag-tagged protein. My ultimate goal is to ChIP for endogenous protein in primary cells after getting the conditions optimized using my cell line.

In brief, I fix ~20x106 cells for 15' w/ 1% formaldhyde at RT, quench and then wash with PBS. I lyse the cells, spin, and then resuspend the nuclei in low salt (200mM) nuclear extract buffer followed by equal volume of a high salt (600mM) NE buffer.

I have no sonication problem as I routine obtain fragment sizes b/t 400 - 1000bp. Since many people seem to struggle with the sonication step, I sonicate my chromatin extract for a total of 60 seconds using a digital Branson sonifier at 60% power in 200uL volume. 1" on/ 1" off x10 pulses, wait 2' on ice and repeat 6 times. The small volume size (200uL) is crucial.

I use 25uL of Invitrogen's M280 strepavidin magnetic beads per IP. I first block the beads with salmon sperm DNA and then preincubate with a biotin anti-rabbit IgG for 4 hours in BSA/PBS, wash w/ BSA/PBS 3x, and incubate with either a rabbit anti-FLAG antibody, rab anti-TF antibody or pre-immune rabbit sera (no antibody control) overnight. Next, I wash and then incubate overnight with my chromatin extract from above at a final concentration of 20mMHepes, 0.5%TritonX, 150mMNaCl, +inhibitors.

The following day I wash the M280 beads with a buffer containing 500mM LiCl2 and 1% TritonX100 6 times for 5' each, followed by TE+ 50mM NaCl 1 time. I then elute with SDS/EDTA for 15' at 65C and reverse cross-links overnight. I phenol/chloroform, EtOH ppt. and perform real-time PCR.

I have the benefit of a positive control gene; someone has demonstrate binding of the TF to a particular gene.

My problem is that I obtain the same fold enrichment for my two negative control regions (one proximal to the positive control gene and another on a different chromosome - insulin promoter) as I do for my positive control region. The signals I obtain for the 'no antibody control' IPs are typically 4-fold weaker (dCp=2) than for the anti-FLAG and anti-TF IPs for each target region. This difference seems too small. The major problem is that both my anti-FLAG IP and anti-TF IP give very strong signals for my positive control region and, unfortunately, for my two negative control regions.

Here is a typical result:
Cp values



Specific Questions:

I pre-bind the antibodies to the M280 magnetic beads instead of first adding the antibody to the chromatin extract and then collecting with the beads. Should this matter? Has anyone compared the two methods?

I tried pre-clearing my chromatin extracts w/ M280 resin, but this resulted in a reduced fold difference for all my samples.

Has anyone used a more stringent washing regime than the 0.5% TritonX / 0.5M LiCl2-containing buffer?

It seems that any anti sera or preimmune sera I use will co-IP DNA. So, I next plan to spike the IP reactions with BSA to block nonspecific binding of my antibodies to the chromatin Is this a good or bad idea? If good, how much BSA should I use? Would it also be wise to spike the chromatin extract with salmon sperm DNA?

If anyone with experience troubleshooting this technique could give me some pointers as to which other steps of my protocol I should vary and test, I would be very appreciative.

Thank you for reading,

-ds

Hi there,
How much antibody are you adding per IP? And how many cells per IP?
We have to use less Ab when we IP fewer cells - otherwise you end up with too much background (and we reduce the amount of beads accordingly).

Clare

Dear Clare,

Thank you so much for your reply. This is something that I was partially mindful of, but only in the sense of trying to maximize the amount of target protein I could IP.

I am using about 5ug of anti-FLAG antibody to IP from 40ug of extract (~2.7x10^7 cells).

If you don't mind, could you tell me what ratio you found to be excessive and normal?

Thanks,

-ds

Hi again

We are looking at histone mods, and have found (in our hands) that 6 million cells per IP is a nice number to work with. Ever since we dropped down to this (from 1x10^7/IP) our ChIPS have been working great (and not to mention easier!).

so for 6 million cells we add:

anti-histone Abs: 1.5ug
IgG: 4.2ug
anti-TF Abs: 4.2ug (note, we haven't tested this in our current system)

I am not sure how much anti-FLAg you should add. Have you done a titration with different amounts of antibody?

Clare

Thank you so much, Clare.

I've not done a titration of the antibody, but that and reducing the number of cells will be the next two things I test.

Thanks again.

-ds

Maybe you should try to use different kind of salts and detergents and not only one, regarding the bead washes (NaCl, LiCl, Triton, Nonidet, Sodium deoxycholate)

hello,

i use always 1million cultured cells per IP and 5ug of antibody in 600ul volume.

Hi again

We are looking at histone mods, and have found (in our hands) that 6 million cells per IP is a nice number to work with. Ever since we dropped down to this (from 1x10^7/IP) our ChIPS have been working great (and not to mention easier!).

so for 6 million cells we add:

anti-histone Abs: 1.5ug
IgG: 4.2ug
anti-TF Abs: 4.2ug (note, we haven't tested this in our current system)

I am not sure how much anti-FLAg you should add. Have you done a titration with different amounts of antibody?

Clare