Periplasmic protein extraction problem! - (Oct/08/2006 )
Dear All,
I worked on extraction of periplasmic protein recently. Actually, this technique have been developed in my laboratory. We keep the cell pellets in the fridge(-20oC) and take out when do the extraction.
Recently, I found the cells were broken after kept in fridge and there were genomic DNA in the supernatant(the periplasmic fraction). These genomic DNA make me a big trouble in extraction.
How can I remove the genomic DNA????
Many Thanks!!!!
siuchi
-siuchi98-
QUOTE (siuchi98 @ Oct 9 2006, 01:17 AM)
Dear All,
I worked on extraction of periplasmic protein recently. Actually, this technique have been developed in my laboratory. We keep the cell pellets in the fridge(-20oC) and take out when do the extraction.
Recently, I found the cells were broken after kept in fridge and there were genomic DNA in the supernatant(the periplasmic fraction). These genomic DNA make me a big trouble in extraction.
How can I remove the genomic DNA????
Many Thanks!!!!
siuchi
I worked on extraction of periplasmic protein recently. Actually, this technique have been developed in my laboratory. We keep the cell pellets in the fridge(-20oC) and take out when do the extraction.
Recently, I found the cells were broken after kept in fridge and there were genomic DNA in the supernatant(the periplasmic fraction). These genomic DNA make me a big trouble in extraction.
How can I remove the genomic DNA????
Many Thanks!!!!
siuchi
one option which i use it to treat the lysed cells with bezonase nuclease from novagen (or any other nuclease eg. mung bean etc etc) which will almost completely degrade the nucleic acids and leave your lysate a little less viscous..
im sure other people will have their favorites also!
enjoy
-Jimmy_september-
QUOTE (Jimmy_september @ Oct 9 2006, 04:33 PM)
QUOTE (siuchi98 @ Oct 9 2006, 01:17 AM)
Dear All,
I worked on extraction of periplasmic protein recently. Actually, this technique have been developed in my laboratory. We keep the cell pellets in the fridge(-20oC) and take out when do the extraction.
Recently, I found the cells were broken after kept in fridge and there were genomic DNA in the supernatant(the periplasmic fraction). These genomic DNA make me a big trouble in extraction.
How can I remove the genomic DNA????
Many Thanks!!!!
siuchi
one option which i use it to treat the lysed cells with bezonase nuclease from novagen (or any other nuclease eg. mung bean etc etc) which will almost completely degrade the nucleic acids and leave your lysate a little less viscous..
im sure other people will have their favorites also!
enjoy
Thank you so much!!!!
One more question, since the extraction and purification of protein should be carry out at 4 oC, I did these two steps in cold room or on ice. Do you think the nuclease work at 4 oC or I should incubate the lysate at 37 oC????? I m afraid of the protein degradation.
many thx
siuchi
-siuchi98-