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Hello Jacob, I am not entirely sure why this has happened to you... A couple of suggestions that may help: 1. dial down the TRIS to 20 mM - you can also use TRIS-HCl (unless the second buffer is there for some other reason). 2. try a 'stronger' resin. I know in the past I've always strongly advocated selectivity over binding strength (i.e. HIS-Select resin over Ni-NTA and its many versions) but in this particular case there may be something weird going on between HIS-Select and Calcium (such as gradual displacement of bound ions, perhaps?). So in this case the use of His-Trap or Ni-NTA etc. may be justified. Let us know how it goes! Artem -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Jacob Keller Sent: Monday, November 16, 2009 3:06 PM To: [log in to unmask] Subject: [ccp4bb] IMAC and Low Ionic Strength Dear Crystallographers, Recently I incubated a small amount of His-select resin with bacterial lysate in (50 mM TRIS-HEPES pH 8.0, 10 mM CaCl2), and washed the resin, for certain experimental reasons, with (50 mM TRIS-HEPES pH 8.0, 0.2 mM CaCl2), at which point a significant amount of my protein fell off the column. The ratio of my protein : background proteins was much higher in these washes, implying that my protein probably had bound, but was now falling off the column due to the 10mM==>0.2mM CaCl2 transition. Has anyone had problems using such low ionic strength wash buffers? Is it possibly necessary for the his-metal-ion interaction? Jacob ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [log in to unmask] *******************************************