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ChIP positive control not working


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#1 jennuottawa

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Posted 19 February 2014 - 07:49 AM

Hi all,

 

I am trying to do ChIP on flash frozen mouse heart tissue. I am using a monoclonal antibody which works in regular IP (for the same flash frozen tissue using teh same buffers and beads) and Western blot, but when I do my ChIP I only get nice products in my input and nothing at all in my IP. I tried doing a ChIP using an RNA pol II antibody as a positive control with GAPDH primers (in coding region- high expressed in QPCR) and have the same problem- input works and .nothing at all in my IP samples... Can anyone give me any tips? I think I must be losing some sample in my IP. 

 

I am using ~ 30mg tissue per I.P.-  minced and fixed in 1.5% PFA 20min

I dounce homogenize the tissue in (25mM tris pH 8.0, 25mM 2-N-morphelino ethane sulfonic acid, 1mM DTT, 0.1% Triton X-100) + protease inhibitor cocktail (Sigma)

when I pellet my nuclei I have a great looking sample under the microscope

I then shear the nuclei and sonicate in lysis buffer- (1% SDS, 10mM EDTA, 50mM Tris-Hcl pH8.1) + protease inhibitors

i use an ultrasonicator so I get one band of 800bp DNA following sonication

I dilute my samples to make 1:5 in ChIP dilution buffer (0.1% SDS, 1.1% triston X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8.1, 167mM NaCl + protease inhibitors) add 5ug of my monoclonal antibody overnight (after a pre-clear with 30ulProtein G agarose beads)

I add 60ul Protein G agarose beads +BSA+ salmon sperm DNA (Millipore) for 1 hour 

i then do washes:

- low salt(0.1% SDS, 1% Triton X, 2mM EDTA, 20mM Tris, 150mM NaCl)

- high salt-0.1% SDS, 1% Triton X, 2mM EDTA, 20mM Tris, 500mM NaCl

- LiC (0.25M LiCl, 1% NP-40, 1% DOC, 1mM EDTA, 10mM Tris)

-2 TE washes for 4 min each

i do 2 consecutive 250ul elutions  for 15min with gentle vortexing (SDS, NaHCO3)and pool them

65oC 4 hours to reverse cross-link with 0.45M NaCl

I then do a proteinase K and RNAse digestion for 1 hour (with EDTA and Tris pH6.5)

I then do a phenol:chloroform:IAA extraction, isopropanol precipitation (with sodium acetate), wash with 70% ethanol,

resuspend DNA in 50uL 10mM tris pH8.0 and use 5uL DNA per PCR reaction

 

I guess my DNA is fine since my input works but something is going wrong in my IP and I am not sure what... I would greatly appreciate any tips I am going crazy! I have attached my full protocol too.

 

Thanks

 

Jenn

 

 

 

 

 

 

Attached Files



#2 memari

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Posted 19 February 2014 - 12:36 PM

I think sonication was too harsh.

Use milder sonication. Time and amplitude.


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Babak Memari

#3 perneseblue

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Posted 02 March 2014 - 10:42 AM

Just curious what class of antibody and from what species are you using?

Protein A does not bind to all class of antibody from all species. It is possible that your antibody is the wrong species type and does not bind to protein A.

 

You need to check. You might need to consider alternatives such as Protein G slurry (protein G doesn't bind to all type of antibodies form all species either), or a bridge antibody between your antibody and protein A, or magnetic beads which are coated with antibodies against your antibody (down side of magbeads is that it is costly and has significantly lower binding capacity).

 

PS:

Do you know if your antibody works on ChIP? Cross linking can sometimes mask the epitope that an antibody binds too. So a simply straight IP is not a certainty that the antibody will work in ChIP.  Try this test experiment, when performing the ChIP, reverse the crosslinks but do not add proteinase K. Then run the eluted protein on Western and check if your protein is pulled down.


May your PCR products be long, your protocols short and your boss on holiday




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