ChIP help- Ab from different sources (View forum version)



Samsuri

Posted 02 August 2011 - 08:26 AM

Hi,

I am attempting to do ChIP for my protein of interest. However i have multiple issues to address and would seek some help. I over express my protein and have tagged it with V5 epitope. (protein is functional and has been tested). I am attempting to IP using antibody for V5 which is conjugated to agarose using the Diagenode kit.

Q1. Is it sufficient to use unconjugated isotype + beads (equal conc. of Ab as V5) as one of the negative controls?

Q2. I use GAPDH promoter (Diagenode kit) primer as negative control. I see an increase in ct for V5-Ab ip compared to IgG, Is that normal?

Q3. I do not know the binding site for my protein to DNA. My hunch is that it may be co-partnering with already known promoter element so i designed primers for it and observed a 1.5 to 2 Ct difference comparing V5 IP to IGG. What difference is considered acceptable?


thanks
Sam

chabraha

Posted 02 August 2011 - 10:33 AM

Q1. Is it sufficient to use unconjugated isotype + beads (equal conc. of Ab as V5) as one of the negative controls?

You can or just beads alone may be better suited......this control, while important, is less so than your GAPDH control

Q2. I use GAPDH promoter (Diagenode kit) primer as negative control. I see an increase in ct for V5-Ab ip compared to IgG, Is that normal?

Depends on the magnitude of increase, but generally in my IPs there is some increase......this is why i subtract out the %Input of my IgG control from the %Input of my IP of interest

Q3. I do not know the binding site for my protein to DNA. My hunch is that it may be co-partnering with already known promoter element so i designed primers for it and observed a 1.5 to 2 Ct difference comparing V5 IP to IGG. What difference is considered acceptable?

It depends on what you feel comfortable with. I think that the more important question is, "what is my fold increase in occupancy of my TF of interest at the site where I think it binds over my negative control region (GAPDH)?".......do this three or so times and then do the statistics

Samsuri

Posted 02 August 2011 - 11:54 AM

ThanKs Chabraha. It was quite helpful.