Stepwise addition to 1% PEI (polyethylenimine) following cell lysis
(before dialysis) should do the trick.
--paul
On 06/25/2015 05:23 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
