ChIP against transcription factors
Posted 13 December 2004 - 09:22 AM
Posted 13 December 2004 - 05:08 PM
I've used CBP from Santa Cruz and POlII CTD, and phos. ser.5 PolII CTD.
I have great results with them (paper is submitted so I can't give you a ref).
Check to see if someone have used thes antibodies in the cells you are using and get those primers. As a general rule, 5 microliters of antibody from upstate,abcam or convance and 10 microliters for santa cruz antibodies.
For example, if you add hormone (estrogen, ret.acid etc) there are published genes you can look at for Pol II, CBP etc. THese results work in many cell lines.
That's about all I can offer.
Posted 13 December 2004 - 06:14 PM
I've done hundreds of ChIPs using abs against acetylated, methylated, ubiquitinated, you-name-it'ed, histones and they work great.
I am also doing ChIP with antibodies to methylated, acetylated histones and have some questions for you.
I have found that loss of DNA during washs after antibody precipitation is a big concern and eventually I could not get an equal signal even for duplicates of samples. What measures do you take to minimize DNA loss and how do you control for that. I know I can use control gene such as beta-actin for acetylated histones, but for transcriptionally silenced genes such as by histone methylation, what gene can be used as control.
I think input DNA can only be used to control for equal amount of starting DNA. Do you agree?
Thank you in advance.
Posted 14 December 2004 - 04:18 AM
Hi, green. As for your questions, I've also done duplicate precipitations and got different strengths of signal. I've tried to use the Abcam protocol which skips the LiCl and TE washes, and I've got stronger signals, but noisier. I believe that the loss of DNA is related to the size of the sonicated fragments. When for some reason I got bad sonications (>5kb) my PCR signals are lower than usual, but when I manage to get better than usual sonications (<1kb) I got high signals, very reproducible and clean.
I agree that input DNA only indicates the amount of STARTING material. What I usually do to account for precipitation efficiency (which may vary from tube to tube, even when using the same ab and the same sample) is analyze my IP'ed samples by duplex PCR. I co-amplify my gene of interest with a positive control (usually beta-actin) which is not suppossed to change. I calculate the amount of problem/control in the sample as a percentage of the problem/control ratio in the input DNA. I usually try to get all my primers to work in duplex with primers for beta-actin so that they more or less amplify at the same efficiency (I use several dilutions of the input DNA to make sure of that).
Hope it helps.
Posted 14 December 2004 - 10:06 AM
If you are studying a gene which is inactivated for example by histone methylation, then it will be hard to find a gene that is similarily silenced as control.
Posted 14 December 2004 - 11:06 AM