Hi Chris, In your Ni-NTA buffer, the total [Na+] could be greater than 530mM after titration. Likely, your protein likes higher concentration salt for surface charge stabilization. Adding Glycerol may further modify the charge distribution and make the protein happy in the solution. For further verification, I usually try the buffer without Glycerin or replace it with small PEG (PEG 100) for +/- impacts, respectively. Indeed, it is pretty good starting point for crystallization.
I noticed that HEPES is also slightly temperature dependent. In most of the cases, the gap could be ignored. It may be helpful. In my previous cases, I tried PBS and Bis-tris with the same pH. The stability was improved great. Can you keep every thing same but different kind of buffer? Hope it would be helpful. Sincerely. On Thu, Jul 13, 2017 at 3:40 PM, Chris Fage <fage...@gmail.com> wrote: > 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 > -- Kevin Jin Sharing knowledge each other is always very joyful...... Website: http://www.jinkai.org/
