Dear Pramod Kumar, if the DNA binds to the protein, wouldn't this affect the interpretation of the Agarose gel?
500 bases should result in a distinct shift during gel filtration. Do you observe this? While waiting for suggestions, you might set up crystallisation trials just in case? Best wishes, Tim On 06/25/2015 11:23 PM, Pramod Kumar 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 > -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A
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