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Personally, I have found that His-SELECT from Sigma appears to have the lowest contaminant binding of the IMAC resins that I've compared. Presumably this is due to the mostly hydrophilic nature of the linker (as compared to mostly hydrophobic linkers of other resins). Note that I have no association with Sigma or any other equipment/supplies manufacturer. As far as flow rates etc. - if you have access to a decent purification workstation (such as any of the AKTA series, or a Biocad [the latter is not supported any more]) - flow rate becomes primarlily dependent on the maximum back pressure recommended by the manufacturer of the resin and the column in which it is poured. Short and fat columns perform better in this regard than long & thin ones, obviously. Highly crosslinked sepharose has decent flow rates and decent pressure characteristics. I routinely run these types of columns at 1 MPa even though manufacturers typically recommend 0.4-0.6 MPa maximum. I believe that Poros media has much higher pressure limit. High-pressure columns tend to be more expensive than lower-pressure ones, of course. If you do not have access to purification equipment capable of medium-pressure chromatography, or if you must resort to multiple gravity-driven columns for parallel work, then batch binding may be an optimum solution. Detergent lysis *without* lysozyme is often better for this kind of work as there's less cell debris generated. Adding DNAse or bensonase to the lysate (even if you French press or sonicate) will reduce your viscosity considerably, as will the extended centrifugation (such as 1hr spin at 20,000 rcf or better). You can 'assist' gravity columns with gentle pressure of air provided that you have liquid in the reservoir so the column never dries up. Artem -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Nat Echols Sent: Sunday, October 08, 2006 2:11 AM To: [email protected] Subject: [ccp4bb]: off-topic: Ni purification media *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** This is indirectly crystallization-related: Can anyone comment on the usefulness of Poros MC resin for purification of His-tagged proteins, especially in comparison to the various Amersham products? I'm dealing with a protein that is happiest with 20% glycerol in the initial buffer, and this makes any FPLC- or pump-based purification very slow due to the relatively low pressure tolerance of Sepharose media. The Poros ion exchange resin works very well and is both robust and cheap, but the MC resin is about 5 times more expensive than anything else I've found. Other than the price, is there any reason not to use it? (For what it's worth, batch purification with Sepharose works relatively well for me, but I've found it too cumbersome in practice and I'm trying to scale up with multiple constructs in this particular case.) thanks, Nat
