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
