Post in EXT] Small scale purifications

Thanks everyone!

Frances

ZRC 1563
Office     646-888-2354
Mobile   516-967-0656


On 8/10/21, 10:22 AM, "Antibody Discussion Forum on behalf of Bover,Laura"
<email obscured> on behalf of <email obscured>> wrote:

    We collect 12 aliquots of 1 ml each and then we measure in nanodrop the OD.
Usually the peak appears in  the 4th-5th elution sample. We pool as need it.
Filter and eventually concentrate.

    Thanks!

    On 8/10/21, 9:14 AM, "Antibody Discussion Forum on behalf of David Blum"
<email obscured> on behalf of <email obscured>> wrote:

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        Frances,

        If you don't want to use an FPLC you can use a simple syringe with the
        pre-packed protein a or g columns and a luer adapter which may be
included
        in the box with the column.  Since this is a bind an elute process it
        should be fine to do it this way, but you would need to determine the
        optimal elution volume and range since you won't have a UV detector.

        On Mon, Aug 9, 2021 at 10:54 AM Weis-Garcia, Frances/Sloan Kettering
        Institute <email obscured>> wrote:

        > Thanks David.
        >
        > Frances
        >
        > ZRC 1563
        > Office     646-888-2354
        > Mobile   516-967-0656
        >
        >
        > On 8/8/21, 1:28 PM, "Antibody Discussion Forum on behalf of David
Blum" <
        > <email obscured> on behalf of <email obscured>> wrote:
        >
        >     Frances:  I understand.  This might not be the best option, but
it
        > should
        >     be good for processing multiple samples quickly without using the
FPLC.
        >     You could also batch bind with the resin you use now and spin
down at a
        >     speed that doesn't deform the beads.  Good luck!
        >
        >     David
        >
        >     On Sat, Aug 7, 2021 at 1:30 PM Weis-Garcia, Frances/Sloan
Kettering
        >     Institute <email obscured>> wrote:
        >
        >     > Thanks David.  The binding capacity is pretty low relative to
        > standard
        >     > resins and the beads are much more expensive.
        >     >
        >     > >    0.6 mg rabbit IgG / ml Pierce™ Protein G Magnetic Beads
        >     >
        >     > while
        >     >
        >     > > 20.0 mg human IgG / ml Cytiva Protein G Sepharose™ 4 Fast
Flow
        > Media
        >     > (formerly GE LifeSciences / Formerly Pharmacia ... boy am I
old)
        >     >
        >     > I know I want to use the FastFlow resin (or something similar)
...
        > right
        >     > now I am more looking for a process that does not involve an
FPLC if
        > I can
        >     > help it.  I have one but the person who has used it is
retireing and
        > this
        >     > is the only thing we will use it for, so do I need it is the
        > question.
        >     >
        >     > A lab (Ivo this was Pop Council) used to grow up a 100s of mls
of
        > each
        >     > hybridoma then line the bottles of conditioned media up on a
shelf,
        >     > allowing the media to flow through a tube into a column below
it and
        > pass
        >     > over the a prot G resin over night by gravity. Some how they
set it
        > up so
        >     > the columns DID NOT DRY OUT.  The next morning they would wash
the
        > columns
        >     > by gravitty again in series and then elute them one at a time.
        > Sounds like
        >     > a great way to do a 5 to 20 at once, and I am sure I could
figure
        > this out,
        >     > but before I spend time on this, was wondering if anyone else
already
        >     > tackled this or had a better mouse trap.
        >     >
        >     > Frances
        >     >
        >     > ZRC 1563
        >     > Office     646-888-2354
        >     > Mobile   516-967-0656
        >     >
        >     > ____________________________________________________________
        >     >
        >     > On 8/6/21, 7:41 PM, "Antibody Discussion Forum on behalf of
David
        > Blum" <
        >     > <email obscured> on behalf of <email obscured>> wrote:
        >     >
        >     >     Have you tried mag beads?  We use them for histagged
proteins at
        > small
        >     >     scale but there are protein coupled beads too.
        >     >
        >     >
https://urldefense.com/v3/__https://www.thermofisher.com/order/catalog/product/88848__;!!PfbeBCCAmug!3WJ0-E0eUzj5cM-MvKLQ5OyZru8t7KhqsTPZP-Gh4ptWUlPQugH9vEtHLaTYxzoe$
        >     >
        >     >
        >     >
        >     >     On Fri, Aug 6, 2021 at 6:47 PM Weis-Garcia, Frances/Sloan
        > Kettering
        >     >     Institute <email obscured>> wrote:
        >     >
        >     >     > Happy Friday.
        >     >     >
        >     >     > Do any of you produce and purify small amounts of the new
MAbs
        > you
        >     > make
        >     >     > for further characterization prior to committing to a few
or
        > one
        >     > winner?
        >     >     > If so, how do you do this.  Currently we make 350 ml
exhausted
        > media
        >     >     > (hybridoma adapted to 1% FBS prescreened for ultra-low
levels
        > of
        >     > bovine IgG
        >     >     > + 1 ng/ml IL6) and then purify it on a Prot G column
using and
        >     > FPLC.  We
        >     >     > get 1 – 8 mg, but I would like to scale this back and
simply
        > it if
        >     > possible
        >     >     > because we usually do not need that much.
        >     >     >
        >     >     > Thanks.
        >     >     >
        >     >     > Frances
        >     >     >
        >     >     >
        >     >     > Frances Weis-Garcia, Ph.D.
        >     >     >
        >     >     > Head
        >     >     > Bi-Institutional Antibody and Bioresource Core Facility
        >     >     > (RRID:  CR 017691)
        >     >     >
        >     >     > Associate Laboratory Member
        >     >     > Immunology Program
        >     >     >
        >     >     > Memorial Sloan Kettering Cancer Center
        >     >     > 1275 York Avenue, New York, NY 10065
        >     >     > <
        >     >
        >
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        >     > >
        >     >     > T 646.888.2354   C 516.967.0656
        >     >     >
        >     >     > ABCF-MSKCC website<
        >     >     >
        >     >
        >
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