Hello all This a follow up of the mail "tips on crystallisation of protein-DNA complex." I would be interested to know about the purity of DNA used for the DNA-complex crystallisation. What does mean by initial screening with the unpurified DNA? Is this mean to start with the desalting DNA rather with HPLC purified ? can any one explain in detail? Iam new to this field. I would highly appreciate the suggestions.
Thanks in advance Peter On Thu, Jul 16, 2009 at 8:54 PM, William G. Scott < wgsc...@chemistry.ucsc.edu> wrote: > Dear Clare et al: > > The absolute classic paper in the field is this: > > Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990). > Crystallization of Escherichia coli catabolite gene > activator protein with its DNA binding site. J. Mol. > Biol. 213, 159–166. > > I've learned more from this than from reading probably any other single > crystallization paper. > > It describes many of the things you ask about, especially trying different > lengths/ends. > > (Last time I had heard from Steve Schultz he had ditched the rat race and > was teaching on the Navajo res. He is my hero.) > > > Bill > > > > > > On Jul 16, 2009, at 7:09 AM, clare stevenson (JIC) wrote: > > I was hoping people could give some tips on the best way to go about >> crystallizing a protein-DNA complex. >> >> I have a large amount of experience in protein crystallisation but have >> never tried co-crystallisation with DNA until I started this project. >> If you want to reply to me personally I will then post a summary. >> >> My protein is a dimer and has been shown by several methods to bind to >> DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have >> several questions: >> >> 1. Do people routinely try different lengths of DNA? >> 2. Do you start with blunt or sticky ends? >> 3. Would purification of the resultant complex by gel filtration be >> a good idea as the interaction is so tight? >> 4. Which screens would you try first? >> 5. Where do you order the DNA from as there is a large difference >> in price depending on supplier. What scale do you go for and what >> purification? >> 6. We expect 1:1 binding. What ratios of DNA to protein are >> generally used (bearing in mind the inaccuracies of protein estimation)? >> >> 7. Any other useful tips? >> >> Thanks in advance for the suggestions and advice >> >> Clare >> >> Dr. Clare E. M. Stevenson >> John Innes Centre, >> Department Biological Chemistry >> Colney Lane >> Norwich >> Norfolk >> NR4 7UH >> >
