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A form of calcium phosphate used in preparative procedures for purification of proteins and nucleic acids in which high flow rate and binding capacity are desired. BSA binding capacity: >12 mg/g; DNA binding capacity: >700 µg/g.
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
12-May-2008 RFH
Synonyms
HA
Description
A form of calcium phosphate used in preparative procedures for purification of proteins and nucleic acids in which high flow rate and binding capacity are desired (BSA binding capacity: >12 mg/g; DNA binding capacity: >700 µg/g).
Form
White solid
Recommended reaction conditions
Hydroxylapatite, Fast Flow ProtocolRe-hydration:
Determine the amount of hydroxylapatite necessary for the intended application. Re-hydrate with 4-6 volumes of starting buffer, such as low ionic strength phosphate buffer, pH >5.5. Gently swirl to thoroughly re-suspend the crystals. Allow the slurry to settle. Decant the excess buffer to approximately give an equal volume of buffer to settled hydroxylapatite. Gently resuspend for a second time in an equal volume of buffer before use.
Packing the Column:
Close the outlet and half-fill the column with starting buffer. Pour the desired amount of fully suspended hydroxylapatite through an attached filling funnel and allow the suspension to settle. After the hydroxylapatite has stabilized, open the column and pour at lease two bed volumes of starting buffer through the column at the appropriate hydrostatic pressure. Prior to the addition of sample, conductivity, pH, or absorbance of the effluent can be used to confirm column equilibration.
Sample Application and Elution:
In general, samples are loaded on the hydroxylapatite in low ionic strength phosphate buffers and are eluted with stepwise or gradient concentration increases. The integrity of the column packing and an accurate measure of the void volume can be determined by applying a small amount of low molecular weight sample which does not bind to hydroxylapatite (e.g. 0.01% methyl orange). This will provide a visual check of uniformity as it passes through the column.
Regeneration of Hydroxylapatite:
High ionic strength phosphate buffer (400-500 mM) is normally sufficient to desorb most material from hydroxylapatite. The use of a high salt solution (1-2 M NaCl), followed by extensive re-equilibration, may help in the removal of contaminants. If necessary, the contaminated top portion of a hydroxylapatite column may be removed, and the column repacked before subsequent use.
CAS number
1306-06-5
Solubility
Phosphate buffer
Storage
+15°C to +30°C
Do Not Freeze
Ok to freeze
Special Instructions
Following reconstitution, store in the refrigerator (4°C).
