Post in EXT] Small scale purifications: Antibody Discussion Forum: ABRF Discussion Forums

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: WARNING: This email originated from outside of MD Anderson. Please validate the sender's email address before clicking on links or attachments as they may not be safe. 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. 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