roger.murphy at ludwig.edu.au
roger.murphy at ludwig.edu.au
Wed Mar 1 21:07:21 EST 2000
When we isolated a FLAG-tagged protein we found we had to either shake the
resin gently overnight with the tissue culture supernatant containing the
protein, then pour into a column, or else recirculate through the pre-packed
column over night. The latter proved easier to do, gave less loss of resin
and protein and resulted in better protein yields. We strched our 25ml column
out to 10 runs but it was definitely "flagging" (couldn't resist!) by the time
we got to number 10.
In article <38BC110D.199621EF at fccc.edu>, gp_adams at fccc.edu wrote:
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>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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>org:Fox Chase Cancer Center;Medical Oncology
>email;internet:gp_adams at fccc.edu
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>fn:Dr. Gregory Adams
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