Bio-Beads®SM Hydrophobic
and Polar Interaction
Adsorbents
Instruction Manual
Bio-Rad Laboratories, 2000 Alfred Nobel Dr., Hercules, CA 94547
LIT195 Rev B
Table of Contents
Section 1 Introduction ..........................................1
Section 2 Product Description..............................2
Section 3 Instructions for Use..............................4
3.1 Preparation of Bio-Beads SM
Adsorbent (Bulk) ............................................4
3.2 Column Method..............................................4
Section 4 Sample Protocols...................................7
4.1 Triton X-100 Detergent Removal...................7
4.2 Adsorption of Unconjugated
Fuorescent Dye ...............................................8
4.3 Batch Protocol.................................................8
4.4 Column Protocol.............................................9
Section 5 Storage.................................................10
Section 6 References............................................10
Section 7 Assistance ............................................12
Section 8 Product Information...........................13
Section 1
Introduction
Bio-Beads SM adsorbents, for adsorbing organics
from aqueous solutions, are neutral, macroporous
polymeric Analytical Grade adsorbents of high surface
area, which are intended for laboratory use only. The
adsorbent is composed of a large number of highly
crosslinked microspheres. This macroreticular structure
gives it high surface area and uniform pore sizes. BioBeads adsorbents can be used with a variety of solvents,
including alcohols, petroleum ether, diethyl ether,
hexane, and solvent mixtures, as well as with aqueous
media. They have excellent physical stability and will
withstand temperatures up to 250 °C and pressures to
1,200 psi. Biotechnology Grade Bio-Beads SM
adsorbents are specifically certified to contain less than
100 microorganisms per gram.
All Bio-Beads SM adsorbents are available in bulk
packages.
1
Table 1. Description of Bio-Beads SM
Adsorbents
Bio-Beads
SM-2
Adsorbent
Chemical nature Polystyrene-divinyl-benzene
Polarity Nonpolar
Dipole moment 0.3 Debye
Average pore diameter 90 Å
(dry beads)
Wet density (g/cc) 1.02
Capacity (Triton
detergent) g/g
®
X-100 0.07
Section 2
Product Description
Bio-Beads SM-2 non-polar polystyrene adsorbents
are particularly useful for the adsorption of nonpolar
substances or surface active agents from aqueous
solutions. Holloway used Bio-Beads adsorbents to
remove Triton X-100 detergent.
the adsorbent for removing Triton,
1
Others have since used
2,3
as well as non-ionic
detergents such as deoxycholate,
8-10
911.
Bio-Beads adsorbents have been used to separate
water soluble steroids,
pesticides,
16-20
trace organics,
4-6
NP-40,7and emulgen
11,12
phenols,13drugs,
21-23
and rhodamine.
24
Table 2 lists some other general applications of Bio-
Beads adsorbents.
Table 2. Compounds Concentrated or
Separated by Bio-Beads SM Adsorbents
Trace organics Morphine
PAH compounds Methaqualone
Hydrocarbons and PCBs Sulfas
Aminocarb insecticides Free rhodamine
Carbamate insecticides Prostaglandins
Ethyl and methyl parathion Steroids
Metals Bile acids
Carboxylic acids Hormones
Phenolic acids Purines and pyrimidines
Flavonoids Acid dyes
Mycotoxins Naringin and limonin
Proline and hydroxyproline Detergents
14,15
2
3
A complete list of more than 100 applications for
Bio-Beads adsorbents can be obtained by requesting
technical bulletin 1461.
Section 3
Instructions for Use
3.1 Preparation of Bio-Beads SM
Adsorbent (Bulk)
Due to the macroporous nature of the adsorbent, air
will sometimes become trapped within the pores. As a
result, it will float in solution. To correct this, slurry the
adsorbent in the solution you will use later. Degas the
slurry by placing it in a vacuum flask and pulling a
vacuum while stirring or sonicating at the same time.
Decant the excess elution solvent. The adsorbent is then
ready for use, either in the batch method or column
method.
3.2 Column Method
1. Slurry the Bio-Beads SM adsorbent in the solvent
that will be used in the process. Usually a 66-75%
4
slurry is necessary to fill the column. Use wet
density information from Table 1 to calculate the
quantity of adsorbent needed for size of the column.
2. Allow the slurry mixture to flow down the side of
the column so that a minimal amount of air bubbles
will be introduced into the chromatographic media
bed.
3. Allow the adsorbent to settle, and elute excess
solvent from the bottom of the column. Make certain
that the solvent level does not go below the top of
the adsorbent bed, because this will allow air bubbles
to form on the top of the column.
4. Equilibrate the Bio-Beads SM adsorbent with 3 bed
volumes of your desired buffer (ex. 10 mM
potassium phosphate, pH 7.2).
5. Apply sample to the column.
6. Wash with 1–2 bed volumes of buffer at 0.3 ml/min.
7. The column can be regenerated by washing in bed
volumes of 100% methanol, followed by rinsing in
DI water.
5
3.3 Batch Method
The batch method is the addition of adsorbent
directly into the sample followed by stirring to achieve
the adsorption. This is a very simple and common
method, though it requires 90–120 minutes, as opposed
to 15 minutes for the column procedure.
1. Weigh 5 g of Bio-Beads SM adsorbent for every 25
ml of solution.
2. Add the adsorbent to the solution.
3. Use either a stir bar or a mechanical agitator to mix
the solution at room temperature for 2 hours.
4. The sample may be recovered by decanting, or by
removing it with a pipette. For smaller samples, it
may be more convenient to centrifuge the sample
before pipetting.
Both columns and batches can be stored. For short
term storage (<1 week), use the elution solvent. For
longer storage, use a 0.05% sodium azide solution or a
20% solution of an organic solvent such as methanol or
isopropanol to prevent microbial growth.
Section 4
Sample Protocols
4.1 Triton X-100 Detergent Removal
Triton X-100 detergent is useful for membrane
solubilization. However, the usefulness of Triton X-100
detergent has been limited by the difficulty experienced
in removing the detergent from the sample. Holloway
reported a simple, rapid, and mild procedure for
removing Triton X-100 detergent on columns of BioBeads SM-2 adsorbents.
on Holloway’s published procedure. Refer to his paper
for complete details.
1. Pack a 1 x 8 cm column (or 1 x 10 cm Econo-
Column
®
chromatography column) with 5 g moist
Bio-Beads SM-2 adsorbent. Equilibrate with 10 mM
potassium phosphate, pH 7.2, at 4 °C. (1 g of BioBeads SM-2 adsorbent will adsorb 0.07 g of Triton
X-100 detergent.)
2. Layer a 1 ml sample of protein solution onto the top
of the bed.
1
The following protocol is based
6
7
3. Wash with 10 mM phosphate buffer at 0.03 ml/min.
Collect fractions and assay for protein.
The column can be regenerated by washing in 4 bed
volumes of methanol, followed by rinsing in DI water.
4.2 Adsorption of Unconjugated
Fluorescent Dye
This procedure was used by Spack et al. to reduce
high background fluorescence with rhodamine-labeled
antibodies.
24
It should be useful for many fluorescent
labels. The Bio-Beads SM-2 adsorbent was prewashed
with 2–3 bed volumes of methanol, distilled water, and
PBS, and stored in PBS until use. If trapped air within
the pores of the Bio-Beads adsorbent causes the
adsorbent to float, the slurried beads may be degassed to
correct this.
4.3 Batch Protocol
1. Add 0.1 g of Bio-Beads adsorbent to 1–2 ml of
solution containing the fluorescent-labeled antibody.
2. Incubate the beads with the solution at room
temperature on a rotary platform to insure thorough
mixing. Stir for 30 minutes.
3. Centrifuge the mixture, and pipette out the
supernatent.
4. Free fluorescent dye will reappear upon storage, so
samples should be adsorbed with the Bio-Beads SM2 adsorbent an hour before use in immunofluorescent
staining experiments.
4.4 Column Protocol
1. Slurry 2 g of Bio-Beads SM-2 adsorbent in
phosphate buffer.
2. Pour a 0.8 x 4 cm Poly-Prep
1 ml bed volume of Bio-Beads adsorbent.
3. Apply 1–2 ml solution containing the fluorescentlabeled antibody, and collect the effluent.
4. Wash the column with PBS and collect the effluent.
®
column containing a
8
9
Section 5
Storage
All Bio-Beads SM adsorbents are stable for 5 years
when stored at room temperature in the unopened
container.
Section 6
References
1. Holloway, P. W., Anal. Biochem., 53, 304 (1973).
2. Bonomi, F. and Kurtz, D. M., Anal. Biochem., 142, 226
(1984).
3. Welling, G. W., Nijmeijer, J. R. J., Van Der Zee, R.,
Groen, G., Wilterdink, J. B. and Welling-Wester, S., J.
Chromatog., 297, 101 (1984).
4. Lorusso, D. J. and Green, F. A., Science, 188, 66 (1975).
5. Shechter, I. and Bloch, K., J. Biol. Chem., 246, 7690
(1979).
6. Garland, R. C. and Cori, C. F., Biochem., 11, 4712 (1972).
7. Momoi, T., Biochem. Biophys. Res. Commun., 87 (2), 541
(1979).
8. Gibson, G. G. and Schenkman, J. B., J. Biol. Chem., 253,
5957 (1978).
9. Warner, M., J. Biol. Chem., 257 (21), 12995 (1982).
10. Bruch, R. C., Thotakura, N. R. and Bahl, O. P., J. Biol.
Chem., 261 (20), 9450 (1986).
11. Shackleton, C. H. L., Sjovall, J. and Wisen, O., Clin.
Chim. Acta, 27, 354 (1970).
12. Bradlow, H. L., Steroids, 11, 265 (1968).
13. Young, C. C. and Tang, C. S., Proc. Nat. Sci. Counc. ROC
(B), 8 (1), 26 (1984).
14. Bastos, M. L., Jukofsky, D. and Mule, S. J., J. Chromatog.,
81, 93 (1973).
15. Digregorio, G. J. and O’Brien, C., J. Chromatog., 101, 424
(1974).
16. Sundaram, K. M. S., Szeto, S. Y. and Hindle, R., J.
Chromatog., 177, 29 (1979).
17. McNeil, E. E. and Otsen, R., J. Chromatog., 132, 277
(1977).
18. Musty, P. R. and Nickless, G., J. Chromatog., 89, 185
(1974).
19. Levesque, D. and Mallet, V. N., Inter. J. Environ. Anal.
Chem., 16, 139 (1983).
10
11
20. Young, C., Proc. Nat. Sci. Counc. ROC (B), 8 (2), 119
(1984).
21. Wigilius, B., Boren, H., Carlberg, G. E., Grimvall, A.,
Lundgren, B. V. and Savenhed, R., J. Chromatog., 391,
169 (1987).
22. Deshmukh, S. W. and Pangarkar, V. G., Indian Chem.
Eng., 26 (3), 35 (1984).
23. Libbey, A. J. Jr., Analyst, 3, 1221 (1986).
24. Spack, E. G., Packard, B., Wier, M. L. and Edidin, M.,
Anal. Biochem., 158, 233 (1986).
25. Joshua, H., Biotechniques, 4 (3), 207 (1986).
26. Lunn, G. and Sansone, E. B., Anal. Biochem., 162, 453
(1987).
Section 7
Assistance
For help in developing your application, contact your
local Bio-Rad representative, or in the U.S., call our
technical service hotline at 1-800-4BIO-RAD. Request
bulletin 1461 for a complete application bibliography.
Section 8
Product Information
Catalog
Number Product Description
152-3920 Bio-Beads SM-2 Adsorbent, 20–50 mesh, 100 g
152-8920 Biotechnology Grade Bio-Beads SM-2
Adsorbent, 20–50 mesh, 25 g
12
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