Flag column

Matt Thomas mthomas at hsc.usf.edu
Mon Feb 28 11:42:07 EST 2000


Currently I'm co-expressing a complex with one of the proteins tagged with
the Flag epitope.  This is done in Baculvirus/Sf9 cells.  After expression,
lysis and a spin, I'm running the lysates over Sgima's Flag resin to clean
it up and as a first step in a purification.  I've found that about 50 to
75% of my protein does not bind and just flows through the column.  I'ved
tried both running it though a column and a batch meathod.  With the batch
working worse than the column.  Anyone have any suggestions on things I
should be careful of using this system?  When I put it on the column my flow
rate is something like 3 to 4 min/ml taking about 30min to load.  I've run
the intial flow through back through with only moderate improvement.  With
the batch meathod I put the resign and lystate in a concial tube and put it
on a rotating wheel in the cold.  

Thanks for any suggestions.  One thing I'm worried about is since I'm
expressing a complex maybe the flag epitope is hidden but since I do get a
reasonable amount bound I'm not convinced of this.


______________________________________________
Matthew Thomas, Ph. D
H. Lee Moffitt Cancer Center, University of South Florida
mthomas at hsc.usf.edu  

"I refrain from publishing for fear that disputes and
controversies may be raised against me by ignoramuses."
   Sir Isaac Newton, correspondence to Liebniz




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