DNA Contaminating My Lysate. - Any way of getting rid of DNA (Sep/27/2005 )
HI,
I am purifying a protein by cooking the lysate at 80 degrees celcius. Its quick and very useful before going on to do a Q-Sepharose column. The problem is that nucleotides or DNA make it impossible to get an A280 reading when I am then eluting from the column. Any quick way of getting rid of this DNA without affecting my protein sample?
Kindest regards,
John
How about using DNase? Sonication on ice will probably shear the DNA, but maybe will affect your protein sample.
Ceri
Ceri
But would that leave me with Nucleotides? I am sonicating already.
Do you spin down your lysates before running them on the column to remove insoluble material?
I usually lyse cells, rest on ice a few mins, then spin at max speed in a microcentrifuge for 10 mins.
This pelllets the insoluble material in the lysate (cytoskeleton etc). I believe it would get rid of some of the DNA/nucleus too. You could then run the supernatant on your column.
If you don't already do this it might be worth a try
Good Luck,
Mountainman
I usually lyse cells, rest on ice a few mins, then spin at max speed in a microcentrifuge for 10 mins.
This pelllets the insoluble material in the lysate (cytoskeleton etc). I believe it would get rid of some of the DNA/nucleus too. You could then run the supernatant on your column.
If you don't already do this it might be worth a try
Good Luck,
Mountainman
I do that already. I also spin down after the heating process. Thanks anyway
There are some DNA binding columns available on market, but I'm not sure how good would be the recovery of your protein.