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.

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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.

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1.3 DNA Grade Bio-Gel HTP

Hydroxyapatite

DNA Grade Bio-Gel HTP hydroxyapatite, supplied in a dry

powder form, has a smaller particle size which significantly
increases its capacity and enhances its selectivity for double­stranded DNA molecules. RNA capacity should also be
increased, making DNA Grade Bio-Gel HTP hydroxyapatite
useful for DNA-RNA hybridization studies. Due to its slower
flow rates, it is recommended for batch chromatography or very
short columns.

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Table 1. Hydroxyapatite Product Performance

Bio-Gel HT Bio-Gel HTP Bio-Gel HTP

Hydroxyapatite Hydroxyapatite Hydroxyapatite

Flow rate cm/h 25-100 35-100 >5
column cross section

Mg BSA sorbed 10 10 10
per dry gram

µg calf thymus DNA 500 500 800
sorbed per dry gram

Storage 4 °C dry form @ dry form @

a

b

room temp. room temp.

hydrated @ 4 °C hydrated @ 4 °C

a. Flow rate determined in a 1.5 x 10 cm column with 40 cm H20

hydrostatic pressure.

b. Batchwise uptake.

c. The DNA capacities listed are lower than previously reported

due to a change in testing methodology. This reporting change
does not represent diminished Hydroxyapatite product prefor­mance or changes to the manufacturing process. The new testing
methodology results in a more accurate determination of DNA
capacity.

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DNA Grade

Section 2
Rehydrating Bio-Gel HTP and
DNA Grade Bio-Gel HTP
Hydroxyapatite

1. Determine the amount of dry powder needed to fill
the column. When hydrated, Bio-Gel HTP hydroxya­patite occupies approximately 2-3 ml per dry gram.

2. Add one part Bio-Gel HTP hydroxyapatite or DNA
Grade HTP hydroxyapatite to six parts of starting
buffer with gentle swirling. Do not use magnetic stir
bars or stirring rods, as these will damage the hydrox­yapatite crystals.

Note: All buffer should be degassed prior to the addition
of the dry hydroxyapatite.

3. Allow the slurry to settle for at least 10 minutes.
Then decant the fines which are in the cloudy upper
level and at the top of the settled bed. Decant to the
settled bed.

6

4. A second decantation is usually not necessary. If it is
desired, add an equal volume of starting buffer to the
bed and mix by swirling gently.

5. Repeat step 3 and resuspend a final time for column
pouring.

Section 3
Resuspending Bio-Gel HT
Hydroxyapatite

1. Bio-Gel HT hydroxyapatite is shipped in 10 mM
sodium phosphate buffer, pH 6.8, containing 0.02%
NaN

. It tends to pack in the bottle during shipping,

3

and requires resuspension for column pouring. If a
buffer other than phosphate is to be used in the actual
elution, it should also be used in the decanting steps.
Pour off the phosphate buffer in the bottle and add
the new buffer solution before suspending the gel.
Buffer pH should remain greater than 6.0.

7

2. Swirl the buffer in the bottle gently until the gel is in
suspension. Do not use stirring rods or magnetic stir
bars.

3. After the gel is suspended, pour it into a beaker and
allow the gel to settle for at least 30 minutes. Decant
the fines which are in the cloudy upper level and at
the top of the settled bed. Decant to the settled bed.

Section 4
Pouring the Column

Many types of columns and bed supports are avail­able. Glass barrel Econo-Column
columns* are useful for hydroxyapatite chromatography.

To pack the column, attach a wide mouth funnel to
the top of the column and add the starting buffer to it.
Then pour the suspended hydroxyapatite into the funnel
and allow 2-3 cm to settle under gravity. Then open the
column outlet, and allow the gel to pack under flow.

* For information on Econo-Column low pressure chromatogra-

phy columns, see Bio-Rad’s current catalog.

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®

chromatography

When the bed is stable, pass at least two bed volumes of
starting buffer through the column.

Section 5
Applying the Sample

A load between 1 and 5 mg of protein per ml bed vol­ume is normally used, although much larger amounts
have sometimes proved satisfactory. Phosphate buffers
are used with a stepwise or gradient increase in concen­tration while the pH is held constant. Generally speaking,
the higher the phosphate concentration the less strongly
the proteins are adsorbed.

Initial experiments are conveniently performed by
adsorbing the protein in 10 mM buffer and eluting the
sample with stepwise concentration increases of about
two-fold. If the effluent is not monitored, at least one and
one-half column volumes of buffer should be used for
each step to provide complete elution of each peak. The
void volume of hydroxyapatite is about 75% of the bed
volume.

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Section 6
Regenerating the Column

A 0.4 M phosphate buffer is usually sufficient to
remove adsorbed materials. If a contaminated sample has
been run, it is advisable to remove the top layer of the
hydroxyapatite bed and wash the remainder of the bed
with one bed volume of 1 M sodium chloride followed
by four column volumes of starting buffer.

For information on Bio-Rad's high performance
hydroxyapatite (Bio-Gel HPHT) columns, request bul­letin 1115, or contact your local Bio-Rad representative.
In the U. S., call technical service at 1-800-4BIORAD.

Section 7
References

21. Stanker, L. D., Vanderlaan, M. and Juarez-Salinas, H., J.

Immunol. Methods, 76, 157 (1985).

22. Brooks, T. and Stevens, A., American Laboratory, 17, 54

(1985).

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23. Juarez-Salinas, H., et al., Methods in Enzymology, 131, 615,

(1986).

24. Juarez-Salinas, H., Brooks, T., Ott, G., Peters, R. and

Stanker, L., Commercial Production of Monoclonal Anti-

bodies, (Seaver, S., ed.) Marcel Dekker, New York.

25. Bukovsky, J. and Kennett, R., Hybridoma, 6, (2) (1987).

26. Salinas, H., et al., BioTechniques, May-June (1984).

27. Smith, G., et al., Anal. Biochem., 141, 432 (1984).

28. Engel, W. D. S., Schagger, H. and Von Jagow, G., Biochim.

Biophys. Acta, 592, 211 (1980).

29. Kock, A. and Luger, T. A., J. Chrom., 296, 293 (1984).

10. Moseman McCoy, M. I., Lubben, T. H. and Gumport, R. I.,

Biochim. Biophys. Acta, 562, 149 (1979).

11. Tsang, A. S. and Coukell, M. B., Eur. J. Biochem., 95, 407

(1979).

12. Gunzburg, J., Part, D., Guiso, N. and Veron, M., Biochem.,

23, 3805 (1984).

13. Kantler, P. M. and Schwartz, H. S., Anal. Biochem., 97, 77

(1979).

14. Beland, F. A., Dooley, K. L. and Casciano, D. A., J. Chro-

matog., 714, 177 (1979).

11

15. Geck, P. and Nasz, I., Anal. Biochem., 135, 264, (1983).

16. Genthner, F., Hook, L. and Strohl, W., Applied and Envi-

ronmental Microbiology, 1007, October 1985.

17. Vincent, lll, W. and Goldstein, E., Anal Biochem., 110, 123,

(1981).

18. Johnson, T. and llan, J., Anal Biochem., 132, 20 (1983).

19. Gorbunoff, M. J., Anal. Biochem., 136, 425 (1984).

20. Gorbunoff, M. J., Anal. Biochem., 136, 433 (1984).

21. Gorbunoff, M. J. and Timasheff, S. N., Anal. Biochem.,

136, 440 (1984).

22. Tiselius, A., Hjertén, S. and Levin, O., Arch. Biochem. Bio-

phys., 65, 132 (1956).

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Section 8
Ordering Information

Catalog
Number Product Description Pkg. Size

130-0150 Bio-Gel HT (Hydrated) 250 ml

130-0151 Bio-Gel HT (Hydrated) 500 ml

130-0420 Bio-Gel HTP (Powder) 100 g

130-0520 DNA Grade Bio-Gel HTP (Powder) 100 g

737-6201 Thermal Chromatography Column, for DNA

Hydroxyapatite

Hydroxyapatite

Hydroxyapatite

Hydroxyapatite

hydroxyapatite chromatography, 1 x 30 cm jack­eted Econo-Column chromatography column and
2 flow adaptors

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