Bio-Rad Bio-Gel HTP Hydroxyapatite User Manual

Bio-Gel®HT

Bio-Gel HTP

DNA Grade Bio-Gel HTP

Hydroxyapatite

Instruction Manual

Table of Contents

Section 1 Properties of Bio-Gel HT, HTP, and

Section 2 Rehydrating Bio-Gel HTP and DNA

Section 3 Resuspending Bio-Gel HT

Section 4 Pouring the Column ............................... 8

Section 5 Applying the Sample .............................. 9

Section 6 Regenerating the Column ...................... 10

Section 7 References................................................ 10

Section 8 Ordering Information ............................ 13

DNA Grade HTP Hydroxyapatite......... 1

1.1 Bio-Gel HT Fully Hydrated Hydroxyapatite.... 3

1.2 Bio-Gel HTP Powder........................................ 3

1.3 DNA Grade Bio-Gel HTP Hydroxyapatite....... 4

Grade Bio-Gel HTP Hydroxyapatite.... 6

Hydroxyapatite ....................................... 7

Section 1
Properties of Bio-Gel HT, HTP,
and DNA Grade HTP
Hydroxyapatite

Hydroxyapatite, a crystalline form of calcium phos­phate, is widely used in preparative biochemistry, having
proven itself a unique tool for the fractionation and
purification of monoclonal antibodies
teins,

8,9

enzymes,

10-12

and nucleic acids.
atite is useful for preparative work in column or batch
modes, and for quantitative analysis of proteins or nucle­ic acids. Its advantages include:

Unique selectivity - Since molecular separation on
hydroxyapatite is not primarily dependent on molecular
weight, molecular size, charge density, or isoelectric
point, hydroxyapatite chromatography is a valuable com­plement to other separations techniques.*

1-7

and other pro-

13-18

Hydroxyap-

* For a discussion of the mechanism of action of hydroxyapatite,

refer to the publications of M. J. Gorbunoff.

1

19-21

High capacity - Hydroxyapatite has a high capacity for
nucleic acids and proteins. Its surface area is about 50 m
per gram.

Low non-specific adsorption-Non-specific adsorption

of hydrophobic substances is minimized by the inorganic
crystalline matrix of hydroxyapatite [Ca

(PO4)3OH]2.

5

Hydroxyapatite displays negligible adsorptive capacity
for low molecular weight substances such as mononu­cleotides, salts, and amino acids.

Chemical and thermal stability - The wide range of
chemical compatibilities (aqueous and inorganic sol­vents), the thermal stability (autoclavable), and the pH
tolerance (pH >5.5) permit the use of hydroxyapatite
under conditions that optimize the binding of nucleic
acids and proteins.

Economy - The initial cost of the material is low, and can
be used several times.

Commercial hydroxyapatite preparations may vary
considerably in their ability to achieve the desired chro­matographic resolution. All Bio-Rad hydroxyapatite is
tested for separation of double-stranded DNA from

2

2

single-stranded DNA, albumin binding capacity, DNA
binding capacity, and flow capacity. The test results for
ds DNA from ss DNA for each batch are printed on the
package label.

1.1 Bio-Gel HT Fully Hydrated

Hydroxyapatite

Bio-Gel HT hydroxyapatite, prepared by the method
of Tiselius,

um phosphate buffer containing 0.02% NaN

et al.,22is shipped suspended in 10 mM sodi-

. This mate-

3

rial gives excellent resolution at a high flow rate (see
Table 1) because of its large particle size. Bio-Gel HT
hydroxyapatite has a shelf life of at least 1 year when
stored at 4 °C in the shipping buffer.

1.2 Bio-Gel HTP Powder

Bio-Gel HTP hydroxyapatite is the Tiselius material
which has been dried by a unique process developed at
Bio-Rad. It may be stored without refrigeration, and,
when resuspended in buffer, it has the same properties as
Bio-Gel HT hydroxyapatite.

3