ChIP - reproducibility problem - (Oct/19/2007 )
I am doing chromatin immunoprecipitation. I am using Upstate kit and protocol and analyzing samples by real-time PCR (SYBR Green). However, the results vary a lot from experiment to experiment. I get PCR product for no antibody control (whashing does not help) and the fold changes which I get for the sample of interest vary from 2 to 10. The same I see for the ChIP positive control, fold change vary from 15 to 25. For second sample of interest the fold change vary from 1 till 3 which mean that some ChIPs are giving positive and another negative results for the same sample. I would appreciate all comments and suggestions. Thank you in advance
I haven't used the Upstate kit but I can offer you an easier, faster, and cheaper protocol. With it I typically can process 24 chromatin samples from setting up the IP to PCR ready DNA in about 6 hours. I get very reprocducible results with it (often having a standard deviation which is less than 10% of the mean). The full protocol is here: Nelson et al., 2006. Nature Protocols 1(1):179-85
If you don't have access to it PM me and I'll send you a reprint.
I use UPSTATE kit with Active motif's magnetic beads.
The descrepency might be due to the residues of insoluble chromatin complexes. I would try centrifuging the chromatin after incubation with antibody and apply only 80% to the protein A beads.
Hope this helps.
The descrepency might be due to the residues of insoluble chromatin complexes. I would try centrifuging the chromatin after incubation with antibody and apply only 80% to the protein A beads.
Hope this helps.
I would agree with newarray, we do this in our own protocol and it is a great source of error if we forget to.
I am experiencing similar problems, and I am really excited to get some answers.
I do have a couple of questions:
1) for how long and at what speed do you centrifuge your samples (after the antibody incubation)?
2) in the short protocol, what is the thought behind incubating your antibody in an ultrasonic bath? Isn't there a risk that you ruin your samples? The levels of my protein of interest is quite low, is it suffient to incubate only for a short time with the antibody in the sonic bath, or du you advice to optimize incubation lenght.
/C
Thank you very much for the advices:))))
Hi,
I also have similar problems as Nikolaj. I use a modified protocol of Upstates and make my own buffers (it' s been 1 year I am doing the same and same ChIP experiments because the results vary in a broad range in the RT-PCR and the 'no-antibody' control gives sometimes strong signal).
I would like to know at which speed and how long one centrifuges the samples before applying to the beads. Maybe this small detail is the solution of my problems(?).
Thanks
I do have a couple of questions:
1) for how long and at what speed do you centrifuge your samples (after the antibody incubation)?
2) in the short protocol, what is the thought behind incubating your antibody in an ultrasonic bath? Isn't there a risk that you ruin your samples? The levels of my protein of interest is quite low, is it suffient to incubate only for a short time with the antibody in the sonic bath, or du you advice to optimize incubation lenght.
/C
After the antibody incubation I centrifuge at 12,000g for 10min (4 degrees C).
The idea behind the ultrasonic bath is that it increases molecular motion to help antibodies find their targets faster. The amount of time required can be optimized. Of the antibodies we've tried the ones that work at all will work with 15-20min in the ultrasonic bath. We haven't tried shorter times but I'm sure some antibodies will work with less than 15min.
I centrifuge at 10,000g for 10 min at 4 degrees. The idea of ultrasonic bath sounds interesting.
The original reference regarding use of ultrasound energy for increasing binding kinetics is here:
http://www.clinchem.org/cgi/content/abstract/30/9/1446