Dear CCP4BB Community, This week, I purified a nicely overexpressing protein by Ni-NTA followed by gel filtration. In a 4 C centrifuge, I concentrated my gel filtration fractions to ~1 mL, transferred the spin filter to ice, and then collected 2 uL for measurement on the Nanodrop. Sadly, the protein precipitated heavily in the pipet tip before I could dispense it onto the Nanodrop pedestal, directly adjacent to my ice box. This effect seems to be abated at 4 C, as the protein remained stable in cold room-chilled pipet tips. However, the protein also precipitated heavily when overnight at 4 C in 1 mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) prior to gel filtration. Has anyone experienced and resolved a similar issue before? Do any useful additives come to mind?
Things I have tried with the gel filtration sample: -Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g. 500 mM). -Exchanging buffer to add 10% glycerol. -Simply diluting the protein in gel filtration buffer to rule out concentration dependence. In each case, the protein precipitates to a milky solution within about a minute of removal from ice (I am working with 20-50 uL volumes in PCR tubes). Many thanks for any suggestions! Best, Chris
