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Even though manufacturers do not recommend this, I routinely use Qiagen's and Sigma's HF IMAC sepharoses under 0.6-1.2 MPa pressure with no apparent ill effects. With heavily particulated or highly viscous loads you will see some shrinking of the column media under high pressure/high flow conditions, however the resin typically just bounces back once high pressure is removed. Naturally, I would not torture a brittle sizing column like that - but with affinity separation one does not need to be as delicate, IMHO. For example: Using a GE (Amersham) XK 26 column, packed with 40 ml of Sigma's HIS-SELECT HF resin, I can run clarified (hard-spun) E. coli lysate at least 10 ml/min on an AKTA Explorer using main system pump to load. This will typically generate total system pressure of 0.5-1.0 MPa depending on lysate viscosity and on how well was the column packed. My high pressure system alarm for these columns is 1.1 MPa (0.65 MPa for XK 50 columns - the fatter they get the less pressure they can take!) With diligent washing, I can re-use these columns dozens of times, with the occasional stripping and regeneration. Frequent treatment with harsh reagents tends to significantly lower the life expectancy of the resin. Please note that this exceeds both the resin AND the column maximum recommended pressures. No guarrantees are offered as to how this will work in your hands. Always protect yourself against column rupture! Artem P.S. outer jackets of the XK columns are VERY sensitive to organic solvents. The manual warns you about this, but it's worth repeating the warning as the hardware is expensive. -----Original Message----- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Nat Echols Sent: Sunday, October 08, 2006 4:46 PM To: ccp4bb@dl.ac.uk Subject: RE: [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 *** > 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. Yikes. The stuff we get from Amersham has pressure limits around 0.3MPa (if that), which is very easy to exceed. (It's lower for gel filtration, but we're used to that.) We have multiple FPLCs - the problem now is I can't really take advantage of them for this step of purification. Part of this is a limited selection of column hardware; the gravity columns I use are significantly wider than any of the casings for FPLC use. If I'm going to spend >$500 on a new casing so I can use higher pressure, I might as well get the pricey media too. > The silica-based Ni-TED resin sold by Macherey-Nagel can take 10MPa > pressure (~1400 psi for the SI-challenged) while the Poros MC resin > takes 17 MPa (~2400 psi). Last time I checked, Ni-TED resin was sold at > about the same price as the 1 MPa Pharmacia Sepharose (Swedish prices). > Make sure that all parts of your purification setup can take these > pressures. I'll look into that media for sure - does it work well? Also, which Sepharose can take 1MPa? I haven't found anything capable of more than 0.3, even the "Fast Flow" columns. thanks, Nat
