STREPTAVIDIN-AGAROSE Product Number S1638 ... - Sigma-Aldrich

STREPTAVIDIN-AGAROSE
Product Number S1638
Storage Temperature 2-8 °C
Product Description
Streptavidin, a 66 kDa homotetrameric protein isolated
from Streptomyces avidinii, is similar to avidin with
respect to its high affinity for biotin (KA ∼ 10 15 M −1 ). 1,2,3,4
Streptavidin is slightly anionic (pI ∼ 5−6) and nonglycosylated,
both properties contributing to the
relatively low non-specific binding compared to egg
white avidin (a glycoprotein with pI ∼ 10.5). 1,2,3,5
Streptavidin is also more resistant than avidin to
dissociation into subunits by guanidinium chloride. 6
Streptavidin-agarose can be used to immobilize or
isolate a variety of biotinylated macromolecules and
complexes (proteins, 7,8,9 antibodies, 3, 9, 10,11,12 lectins, 9,13
nucleic acids, 9,14,15,16 receptors and ligands 9,14 ). The
binding of biotinylated macromolecules is essentially
irreversible because of the harsh conditions needed to
disrupt the streptavidin-biotin interaction. This feature
makes streptavidin-agarose useful in a variety of affinity
purification applications. 9,14
This streptavidin-agarose conjugate is prepared by
attaching streptavidin (S 4762) to a cyanogen bromideactivated
4% agarose (A 6763) through an amino
group, resulting in a 7-atom spacer. 17
Reagents
Streptavidin-agarose is provided as a suspension in
0.01 M sodium phosphate, pH 7.2, containing 0.15 M
NaCl and 0.02% sodium azide.
Precautions and Disclaimer
Streptavidin-agarose is for laboratory use only; not for
drug, household or other uses. The storage buffer
contains 0.02% sodium azide which is toxic. Please
refer to the Material Safety Data Sheet (MSDS).
Storage/Stability
The suspension should be stable approximately two
years stored at 2-8 °C. DO NOT FREEZE. Freezing
will damage bead structure.
Preparation Instructions
The agarose beads must be washed thoroughly with 5
to 10 column volumes of buffer to remove the sodium
azide in the storage buffer.
Sample Procedures
Note: These procedures are provided as general
guidelines. Appropriate and optimal conditions should
be determined empirically by the end user for any
specific application.
Procedure for Column Purification of Biotinylated
Proteins
1. Pour the streptavidin-agarose into an appropriate
column and wash with 5 to 10 column volumes of
PBS.
2. Apply the sample containing the biotinylated
protein.
3. Wash with PBS until the absorbance at 280 nm is
minimal.
4. Elute biotinylated proteins using 6 M guanidine HCl,
pH 1.5-2 or by boiling in 2% SDS with 0.4 M
urea. 9,17
5. Immediately dialyze or desalt eluted samples if
desired.
Procedure for Immunoaffinity Column Purification of a
Protein 3
1. Pour the streptavidin-agarose into an appropriate
column and wash with 5 to 10 column volumes of
PBS.
2. Apply the biotinylated antibody.
3. Wash the column with PBS until the absorbance at
280 nm is less than 0.01-0.02.
4. Apply the sample (antigen) to the column.
5. Wash with PBS until the absorbance at 280 nm is
minimal.
6. Elute the sample (antigen) with 0.1 M acetic acid or
0.1 M glycine HCl (pH 2.5) or other elution buffer to
dissociate the antibody-antigen interaction. 3,9,16,19
Immediately neutralize eluted samples.
Notes:
a) Removal of biotinylated substances from
streptavidin-agarose: In order to avoid the harsh
and denaturing conditions required to dissociate the

iotin-streptavidin interaction, consider the use of
monomeric avidin-agarose (A 2036) which requires
milder dissociation conditions, such as the use of
free biotin or low pH. 20 Another option is to
iminobiotinyate the protein of interest.
Iminobiotin has a pH-dependent interaction with
streptavidin which allows tight binding at pH > 9.5
and dissociation at pH 4. 21,22 For biotinylated
nucleic acid probes, 2-mercaptoethanol or DTT can
reportedly be used to disrupt the streptavidin-biotin
interaction. 15
b) Binding inhibitors: Non-dialyzed, nonfat dried milk,
which may contain biotin 23 and various sugars
(such as mannose), 24 is reported to interfere with
the binding of biotin to streptavidin and avidin.
c) Reduction of non-specific binding: If non-specific
binding is a problem, consider the following:
i. Use a different buffer system or increase the
concentration of NaCl to 0.5 M. 25
ii. Increase detergent concentration or use a
different detergent.
iii. Use a cleared cell lysate (centrifuge the cell
lysate), to remove unwanted components (see
procedure in reference 16).
iv. Wash the resin to remove non-specifically
bound proteins (such as a high pH buffer wash
(borate buffer, pH 8-9) 25 , followed with a low
pH buffer (acetate buffer, pH 4)).
v. Consider using a product with a different
charge, such as avidin-agarose or monomeric
avidin-agarose.
References
1. Chaiet, I. and Wolf, F.J. The properties of streptavidin,
a biotin-binding protein produced by Streptomyces.
Arch. Biochem. Biophys. 106: 1-5 (1964).
2. Bayer, E.A., Ben-Hur, H. and Wilchek, M., Isolation
and properties of streptavidin, Meth. Enzymol., 184:
80-89 (1990).
3. Bayer, E.A. and Wilchek, M. Avidin-Biotin
Technology, Methods in Molecular Biology, Vol. 10:
Immunochemical Protocols, p. 137-162 (1992).
4. Argaraña C.E. et al., Molecular cloning and
nucleotide sequence of the streptavidin gene,
Nucleic Acids Res.14(4):1871-1882 (1986).
5. Green, M.N. Avidin and Streptavidin, Meth.
Enzymol, 184: 51-67 (1990).
6. Green, N., Avidin, Advances in Protein Chem., 29:
85 (1975).
7. Swack J.A. et al., Use of avidin-sepharose to
isolate and identify biotin polypeptides from crude
extracts, Anal. Biochem. 87(1): 114-26 (1978).
8. von Boxberg, Y. et al. Use of the biotin-avidin
system for labelling, isolation and characterization
of neural cell-surface proteins. Eur. J. Biochem.
190 (2): 249-256 (1990).
9. Wilchek, M. and Bayer, E.A. Applications of Avidin-
Biotin Technology: Literature Survey, Meth.
Enzymol., 184: 14-45 (1990).
10. Updyke, T.V. and Nicolson, G.L., Immunoaffinity
isolation of membrane antigens with biotinylated
monoclonal antibodies and streptavidin-agarose.
Methods Enzymol.121:717-25 (1986).
11. Updyke, T.V. and Nicolson, G.L., Immunoaffinity
isolation of membrane antigens with biotinylated
monoclonal antibodies and immobilized streptavidin
matrices. J. Immunol. Methods 73(1):83-95 (1984).
12. Gretch, D.R. and Stinski, M.F. The use of
biotinylated monoclonal antibodies and streptavidin
affinity chromatography to isolate herpesvirus
hydrophobic proteins or glycoproteins, Anal.
Biochem. 163: 270-277 (1987).
13. Buckie, J.W. and Cook, G.M., Specific isolation of
surface glycoproteins from intact cells by
biotinylated concanavalin A and immobilized
streptavidin. Anal. Biochem. 156(2):463-72 (1986).
14. Diamandis, E.P. and Christopoulos, T.K. The
Biotin-(Strept)Avidin System: Principles and
Applications in Biotechnology, Clin. Chem. 37: 625-
636 (1991).
15. Jenne, A. and Famulok, M. Disruption of the
Streptavidin Interaction with Biotinylated Nucleic
Acid Probes by 2-Mercaptoethanol, BioTechniques
26:249-254 (1999).
16. Ausubel, F.M., et al. Eds., Short Protocols in Molecular
Biology, 4th edition, John Wiley & Sons, Inc. p.
10-89, 12-28,17-5, 1999 (Product No. Z36,436-3).
17. Sigma production/quality control.
18. Assay method based on Green, N., Meth.
Enzymol., XVIIIA, 418 (1970).
19. Harlow, E. and Lane, D. Antibodies: A Laboratory
Manual. Cold Spring Harbor Laboratory Press,
New York, p. 549 (1988).
20. Green, N.M. and Toms, E.J. The properties of
subunits of avidin coupled to sepharose. Biochem.
J. 133(4): 687-700 (1973).
21. Heney G. and Orr G.A., The purification of avidin
and its derivatives on 2-iminobiotin-6-aminohexyl-
Sepharose 4B, Anal. Biochem. 114(1): 92-6 (1981).

22. Orr, G.A. The use of the 2-iminobiotin-avidin
interaction for the selective retrieval of labeled
plasma membrane components, J. Biol. Chem.
256: 761-766 (1981).
23. Hoffman, W.L. and Jump. A.A., "Inhibition of the
Streptavidin-Biotin Interaction by Milk", Anal.
Biochem., 181, 318 (1989).
24. Houen, G. and Hansen, K. Interference of sugars
with the binding of biotin to streptavidin and avidin.
J. Immunol. Methods 210(2): 115-123 (1997).
25. Duhamel, R.C. and Whitehead, J.S. Prevention of
Nonspecific binding of avi din, Meth. Enzymol., 184:
201-207 (1990).
kat 11/99
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