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
