TF crosslinking in mitochondrial genome - (Jan/30/2007 )
Hi all.
I'm trying to develop the crosslinking of a TF of the mtDNA of rat liver cells to validate previous results obtained by EMSA. I have tryed to use the first part of the protocol done by Kaufman et al. (PNAS.2000;97(14)) adapted to hepatocytes and then trying to do the crosslinking and IP using the Upstate protocol. After several months I have good results, but now I have problems of reproducibility. One of my big problems is the low yield of the mtDNA after the IP. Another problem is the small size and complexity of the mtDNA compared with nuclear DNA. Has anybody experience in "CHIP" in the mtDNA? Do you know any best protocol that I can use? How can I increase the compexity of the mtDNA (nuclear, plasmid....????)?
Is it possible to do in mitochondria?????
Thank you very much.
I am not too sure if mtDNA is packaged the same way as nucDNA very interesting to see if it is so.
Miguel, Is there a way you can enrich for mitochondria? I am thinking something along the lines of a dunce homogeniser that will leave only intact mitochondria, that way you will have an enrich sample of it and remove all the nuclei and other rubbish from it.
Nick
Miguel, Is there a way you can enrich for mitochondria? I am thinking something along the lines of a dunce homogeniser that will leave only intact mitochondria, that way you will have an enrich sample of it and remove all the nuclei and other rubbish from it.
Nick
Dear Nick. In fact I'm working with enriched mitochondria (mitochondria isolated in sucrose gradient) and i treat my intact mitochondria with Dnase to avoid nuclear contamination, then I add the formaldehyde and after adding glycine and several washes I lyse the organelles with NP-40. After that, I sonicate and I develop the IP UPSTATE protocol. I'm working with a final mito concentration of 2 mg/ml and at the end i get poor yield of DNA (5-10 mg/ml) I'm worried of incresing the mito concentration due to the background, but I have problems of reproducibility ( in general i have good results with the inespecific Ab and the no Ab. The problem is that sometines i have the specific signal and when I try to replicate my experiment the signal disapear......)
The mitoDNA is a nude, small DNA (16,5 kb) with no histones, circular. But it has a TF called Tfam that binds to the regulatory region of mtDNA (D-loop) but also to a lot of sequences of the mtDNA and it has benn hipothesized that it could act as and histone-like protein for mtDNA. In fact I use an Ab anti-Tfam as a possitive control. I'm trying to study anotjer TF of the mtDNA.
Any suggestions?
Thank you very much
Miguel, Is there a way you can enrich for mitochondria? I am thinking something along the lines of a dunce homogeniser that will leave only intact mitochondria, that way you will have an enrich sample of it and remove all the nuclei and other rubbish from it.
Nick
Dear Nick. In fact I'm working with enriched mitochondria (mitochondria isolated in sucrose gradient) and i treat my intact mitochondria with Dnase to avoid nuclear contamination, then I add the formaldehyde and after adding glycine and several washes I lyse the organelles with NP-40. After that, I sonicate and I develop the IP UPSTATE protocol. I'm working with a final mito concentration of 2 mg/ml and at the end i get poor yield of DNA (5-10 mg/ml) I'm worried of incresing the mito concentration due to the background, but I have problems of reproducibility ( in general i have good results with the inespecific Ab and the no Ab. The problem is that sometines i have the specific signal and when I try to replicate my experiment the signal disapear......)
The mitoDNA is a nude, small DNA (16,5 kb) with no histones, circular. But it has a TF called Tfam that binds to the regulatory region of mtDNA (D-loop) but also to a lot of sequences of the mtDNA and it has benn hipothesized that it could act as and histone-like protein for mtDNA. In fact I use an Ab anti-Tfam as a possitive control. I'm trying to study anotjer TF of the mtDNA.
Any suggestions?
Thank you very much
You may try optimizing the crosslinking. If your TF doesn't bind tightly to the mtDNA then it may not be getting crosslinked efficiently. You might try increasing the crosslinking time to 15 min and the formaldehyde concentration to 1.5%. Also, others have used paraformaldehyde since it can crosslink over larger distances.