Separating DNA and nucleotides from protein - (Feb/19/2006 )
IN BREAKING DOWN MY CELLS AND PURIFYING MY PROTEIN, I HAVE ALSO BROKEN DOWN DNA SIGNIGICANTLY AND ITS ALL OVER MY PURIFIED PROTEIN AFTER GEL FILTRATION. THE SOLN ACTUALLY LOOKS BROWN. MY NEXT TWO STEPS ARE ION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY AND I DONT WANT IT GETTING IN THE WAY. CAN ANYONE THINK OF A WAY OF GETTING RID OF THIS A260 PEAK.
JBC
-johncarrigan-
QUOTE (johncarrigan @ Feb 19 2006, 12:26 PM)
IN BREAKING DOWN MY CELLS AND PURIFYING MY PROTEIN, I HAVE ALSO BROKEN DOWN DNA SIGNIGICANTLY AND ITS ALL OVER MY PURIFIED PROTEIN AFTER GEL FILTRATION. THE SOLN ACTUALLY LOOKS BROWN. MY NEXT TWO STEPS ARE ION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY AND I DONT WANT IT GETTING IN THE WAY. CAN ANYONE THINK OF A WAY OF GETTING RID OF THIS A260 PEAK.
JBC
JBC
Hi, to eliminate nucleic acids I usually used streptomycin also you can used protamin (these reagents can precipitate nucleic acids). I usually add directly the antibiotic in my extract also you can meke a solution, stirring at 4C during 20 min, and then centrifuge at 13,000 rpm, this procedure is recomended by enzymology book.
-operon-
QUOTE (operon @ Feb 19 2006, 07:22 PM)
QUOTE (johncarrigan @ Feb 19 2006, 12:26 PM)
IN BREAKING DOWN MY CELLS AND PURIFYING MY PROTEIN, I HAVE ALSO BROKEN DOWN DNA SIGNIGICANTLY AND ITS ALL OVER MY PURIFIED PROTEIN AFTER GEL FILTRATION. THE SOLN ACTUALLY LOOKS BROWN. MY NEXT TWO STEPS ARE ION EXCHANGE AND REVERSE PHASE CHROMATOGRAPHY AND I DONT WANT IT GETTING IN THE WAY. CAN ANYONE THINK OF A WAY OF GETTING RID OF THIS A260 PEAK.
JBC
Hi, to eliminate nucleic acids I usually used streptomycin also you can used protamin (these reagents can precipitate nucleic acids). I usually add directly the antibiotic in my extract also you can meke a solution, stirring at 4C during 20 min, and then centrifuge at 13,000 rpm, this procedure is recomended by enzymology book.
Well thanks. That sounds like just the ticket. What concentration of streptomycin are we thinking about here?? Thanks for your help again. I really appreciate it.
jc
-johncarrigan-
Actually, Ive been told to use 1% so thats ok.
The only problem now is that the next stage of the purification of Q-Sepharose and this will surely bind the streptomycin sulfate wont it? Perhaps I might post this up as a question.
-johncarrigan-
QUOTE (johncarrigan @ Feb 20 2006, 09:23 AM)
Actually, Ive been told to use 1% so thats ok.
The only problem now is that the next stage of the purification of Q-Sepharose and this will surely bind the streptomycin sulfate wont it? Perhaps I might post this up as a question.
The only problem now is that the next stage of the purification of Q-Sepharose and this will surely bind the streptomycin sulfate wont it? Perhaps I might post this up as a question.
What about degrading DNA completely?
- and maybe dephosphorylate nts if that can make separation easier
Shrimp nuclease works well at temperatures compatible with proteins, and has very high spec activity, allowing for use of minor amount of enzyme.
SAP of cause takes care of dephosph, if necessary.
-Gerd-