Several different approaches may help you to separate DNA from protein including;
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. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase "breathing") to disrupt the interaction. 3) Combine salt with low concentrations of denaturants. 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. Dan Daniel A. Bonsor PhD Institute of Human Virology, University of Maryland at Baltimore 725 W. Lombard Street N571 Baltimore MD 21201 ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar [pramod...@gmail.com] Sent: Thursday, June 25, 2015 5:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Interesting DNA contamination Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
