pramod Do a spin after bacterial cell breaking at 8K to get rid of a nuclear pellet Also there is a nuclease preparation available called benzonase it is most effective when you also add magnesium in your buffer so in your homogenization buffer add magnesium and should avoid any EDTA Pius
On Thu, Jun 25, 2015 at 2:23 PM, Pramod Kumar <pramod...@gmail.com> wrote: > 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 > -- P
