Bio-Rad Media Sampler Pack - Manual (Page 9)

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Section 6. Storage
Recommended storage of CFT is in 0.1–1 M sodium or potassium hydroxide at
room temperature. It can also be stored in low concentrations of sodium or
potassium phosphate buffer (pH 6.8) with 20% (v/v) ethanol or methanol.
Unused CFT should be stored in its original sealed container at room temperature.
Section 7. Tips and General Information
1. Elution buffers — For most applications, elute bound fractions with linear or
step gradients of potassium or sodium phosphate at neutral pH (6.8–7.2),
going from low (5–10 mM) to high (400 mM) phosphate buffer concentrations.
Use potassium phosphate when operating at temperatures below 12°C
because sodium phosphate can precipitate at the lower temperatures.
2. Method optimization — Binding capacity and retention time decrease with
increasing pH. CFT ceramic fluoroapatite is a calcium phosphate compound
that dissolves at low pH. When used at pH 4, the CFT degrades quickly. After
100 cycles in pH 5 phosphate buffer, there is a small loss in mass of CFT. After
100 cycles in pH 5.5 phosphate buffer, there is no noticeable loss in mass.
Loading can be performed in low concentrations of phosphate (10–20 mM)
between pH 5.5 and 6.8, and elution can be done with linear or step gradients
at higher phosphate concentrations (up to ~400 mM).
3. Other buffers — Special attention to selection of the buffer system is
recommended when deviating from standard phosphate buffer because the
stability of CFT may be adversely affected. Chelating buffers, such as PIPES,
should not be used with CFT.
4. Chelating agents — Do not use EDTA or other chelating agents in the
sample or the buffers. Chelating agents bind to the calcium in the CFT and
eventually dissolve the support.
5. Sodium chloride — Basic proteins (pl > 7) elute with NaCl, whereas acidic
proteins (pl < 7) do not.
6. Calcium carbonate — Carbonate-free water should be used to prepare all
solutions. Carbonate reacts with calcium ions to form a crust of calcium
carbonate at the top of the column. The severity or rate of carbonate buildup
on CFT is dependent on the quality and volume of water used.
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