Affinity Chromatography - Department of Molecular and Cellular ...

The harsh conditions required to break the streptavidin-biotin bond may affect both the
sample and the ligand. Streptavidin Sepharose columns cannot be re-used after elution
under these conditions.
Antigen purification
Antigens can be purified from biotinylated antibody-antigen complexes bound to streptavidin.
The following method was adapted for HiTrap Streptavidin HP from work published in
Anal. Biochem. 163, 270–277 (1987), Gretch, D.R., Suter, M. and Stinski, M.F.
Solubilization buffer: 20 mM sodium phosphate, 150 mM NaCl, pH 7.5 with 0.1% SDS, 1.0% Nonidet-P-40,
0.5% sodium deoxycholate, 0.02% NaN 3 , 100 µg/ml PMSF
Elution buffer: 0.1 M glycine-HCl, pH 2.2
1. Solubilize the antigen with an appropriate amount of solubilization buffer, clear the sample by centrifuging at
12 000 g for 15 min.
2. Add the biotinylated antibody and adjust the volume to 1 ml.
3. Incubate with end-over-end mixing, for at least 1 h or overnight.
4. Equilibrate the column with 10 column volumes of solubilization buffer.
6. Apply antibody-antigen solution to the column at a low flow rate such as 0.2 ml/min. If the sample volume is
less than 1 ml, apply the sample, and leave for a few minutes to allow binding to take place.
7. Wash out unbound sample with 10 column volumes of solubilization buffer or until no material is found in
eluent (monitored by UV absorption at A 280 nm ).
8. Elute with 5–10 column volumes of elution buffer.*
*Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (100 µl – 200 µl
1 M Tris-HCl, pH 9.0 per ml fraction), so that the final pH of the fractions will be approximately neutral or perform a
rapid buffer exchange on a desalting column (see page 134).
Media characteristics
Composition pH stability* Mean particle size
Streptavidin Sepharose Streptavidin is coupled Short term 2–10.5 34 µm
High Performance to Sepharose High Long term 4–9
Performance using a
HiTrap Streptavidin HP N-hydroxysuccinimide
coupling method.
*Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects
on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place
and sanitization procedures.
Storage
Wash media and columns with 20% ethanol (use approximately 5 column volumes for
packed media) and store at +4 to +8 °C.
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