4 replies to this topic

[vecttoraz]

Dear collegues,

I am trying to prove protein A binds to my promoter region. Had good sonication of my cell and clear results using anti-histone H3 antibody. I am doing analysis by common PCR. When using my specific antibody (good one from abcam), I see that I have a stronger band using the specific antibody as compared to using rabbit IgG ou no antibody for IP. My PCR gel is attached.

I will be very gratefull if somebody could help me looking at my results and suggesting, because I have no experience.

thanks in advance

vecttoraz  

Attached Files

•  CHIP.pdf   30.31K   136 downloads

[chabraha]

Just a guess here but it looks like your doing endpoint PCR with too many cycles........I would highly recommend using real-time PCR. You could use densitometry to determine differences in the intensity of your bands, but I'd be willing to bet you'd get a different result every time..........By my eye, and under the current conditions, I'd say that you have minimal enrichment over your Rabbit IgG.......and around the same enrichment as your %Input.

[vecttoraz]

Dear Enthusiast,
thank you very much for your reply. I will repeat my PCR with less cicles. hope that can help me.
best wishes

[Mighty Mouse]

vecttoraz, on 03 June 2011 - 03:37 AM, said:

Dear Enthusiast,
thank you very much for your reply. I will repeat my PCR with less cicles. hope that can help me.
best wishes

I agree with Chabara...keep in mind you want to be about the middle of your exponential amplification phase in your PCR, otherwise you can't tell much of anything. Would be worth titrating your cycles to figure out what work sbest.

MM

[vecttoraz]

I will be doing real-time pcr!!! thank you all!!!