Hi Thanks all, I appreciate all the valuable inputs..
Piush.. I ll be trying Benzonase up next.. but since the DNA appears so secluded for DNAses, it makes me little skeptical as mg and DNAse already been there for ON dialysis. ............ Tim if the DNA binds to the protein, wouldn't this affect the interpretation of the Agarose gel? I can send you the Ni-elution SDS pic where the GFP taged protein and DNA band appers at two different different and distinct sizes. 500 bases should result in a distinct shift during gel filtration. Do you observe this? Since I don't have free protein to compare with DNA-bound, m not able to see the shift of peak. While waiting for suggestions, you might set up crystallization trials just in case? Yea, just did that :) .................. Tom Why not crystallize the whole complex? It would be more interesting than the protein alone- higher impact and all that. kept the trays, wish me good luck :) ................ Dan 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. Yes, Its nice piece of info. Just one concern eats me, that is the longer wash that I observed some time with mem-proteins may lead to release of native bound lipids and causes deterioration. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase "breathing") to disrupt the interaction. I ll be doing that provided it tolerate. 3) Combine salt with low concentrations of denaturants. Incorporated to optimization list 4) Try a couple of different restriction enzymes such as DpnI or FatI to see if you can break it into smaller fragments. If you can, you maybe able to clone into a cloning vector with compatable ends/blunt ligation to sequence and identify the region of host DNA that is causing the problem. Excellent input, ll be doing. ....................... paul Stepwise addition to 1% PEI (polyethylenimine) following cell lysis (before dialysis) should do the trick. You mean, membrane and protein extraction as well Ni-Aff in presence of ~1% PEI? ................... Katharina Since your protein apparently binds ATP – do you know the active site? Maybe its worthwhile to do an active site mutant (or any other possible DNA binding mutant?) and to see if it makes any difference in DNA binding? Nice input, Working on it. Another question is, if the DNA you observe is somehow an homogeneous species. Yes, consistantly Otherwise, maybe there’s a way to somehow digest the DNA further, in presence of the protein, after purification to obtain an homogeneous species, than to get rid of the nucleases again and than to simply set up crystallization trays. To set up crystalliyation trials I would do anyways, in case you have enough protein. Kept Trays, :) Thanks again :) Pramod
