Performing a Separation of Biotin and Biotinylated Substances with HiTrap Streptavidin HP and Streptavidin Sepharose High Performance

Biotin and biotinylated substances bind to streptavidin (a molecule isolated from Streptomyces avidinii) in a very strong interaction that requires denaturing conditions for elution. By coupling streptavidin to Sepharose, a highly specific affinity medium is created and, using biotinylated antibodies, the strong interaction can be utilized for the purification of antigens. The biotinylated antibody-antigen complexes bind tightly to Streptavidin Sepharose and the antigen can then be eluted separately using milder elution conditions, leaving behind the biotinylated antibody. An alternative to labelling the antibody with biotin is to use 2-iminobiotin that binds to streptavidin above pH 9.5 and can be eluted at pH 4 (Figure 40).

Purification Options

* See Appendix 4  to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Performing a separation:

Biotinylated substances

Binding buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5

Elution buffer: 8 M guanidine-HCl, pH 1.5

Iminobiotinylated substances

Binding buffer: 50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0

Elution buffer: 50 mM ammonium acetate, 0.5 M NaCl, pH 4.0

1. Equilibrate the column with 10 column volumes of binding buffer.
2. Apply the sample. For the best results use a low flow rate, 0.1–0.5 mL/min, during sample application.
3. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm).
4. Elute with 10–20 column volumes of elution buffer.*

* Since elution conditions can be 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 133, Column packing and preparation).

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% NaN₃, 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.
5. 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.
6. 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}).
7. 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 133, Column packing and preparation).

Media Characteristics

* 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.