Gregory P. Adams, Ph.D.
gp_adams at fccc.edu
Tue Feb 29 13:33:54 EST 2000
We have experienced similar problems to different extents depending upon the
protein we expressed. In general, we have found that passing the flow through
through the column does aid in the isolation of more protein. However, there
are a few quick issues to consider. First, the lifespan of these columns in
not great (maybe 4-5 uses before a new rather expensive column is required).
So, if the protein is really important, a new column is suggested. Second, I
believe that you may be right that the tag may be hidden (I think that this
happens to us sometimes even when expressing a single protein). Have you
checked the flow through on a western blot (anti-Flag) to determine if the tag
is present on the protein that was not retained? If you have specific antisera
to your protein you could also check to make sure that the flow through is
really the protein that you are after.
Matt Thomas wrote:
> 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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