Protein Protocols Protein Protocols

Preparation of Avidin Conjugates 369
Table 1
Molar Extinction Coefficients at 280 nm and Molecular Weights of Avidin
and Avidin-Like Proteins
Protein Molecular weight EM Avidin/cm/M
Avidin 66,000 101,640
Deglycosylated
avidin/NeutrAvidin 60,000 101,640
CaptAvidin biotin-binding protein 66,000 118,800
Streptavidin 60,000 180,000
4. Continue stirring at room temperature for 1 h.
5. Dialyze the solution in 2 L of 0.1 M phosphate buffer at 4°C for 24 h, with four buffer
changes using a membrane with a suitable molecular weight cutoff.
3.1.3. Deprotection of the Avidin Thiol Groups
This procedure is carried out immediately before reacting thiolated avidin with the
maleimide derivative of the antibody or enzyme prepared in Subheading 3.1.2.
1. Dissolve 3 mg of TCEP in 0.3 mL of 0.1 M phosphate buffer.
2. Add 11 µL of TCEP solution to the thiolated avidin solution. Incubate for 15 min at room
temperature.
3.1.4. Formation and Purification of the Conjugate
1. Add the thiolated avidin–TCEP mixture dropwise to the dialyzed maleimide derivatized
protein solution with stirring. Continue stirring for 1 h at room temperature, followed by
stirring overnight at 4°C.
2. Stop the conjugation reaction by capping residual sulfhydryls with the addition of NEM at
a final concentration of 50 µM. Dissolve 6 mg of NEM in 1 mL of DMSO and dilute
1:1000 in the conjugate reaction mixture. Incubate for 30 min at room temperature or
overnight at 4°C (see Note 8). The conjugate is now ready for final purification.
3. Concentrate the avidin–protein conjugate mixture to 1–2 mL in a Centricon ® -30 (Amicon,
Beverly, MA) or equivalent centrifuge tube concentrator.
4. Pack appropriate size columns (e.g., 10 × 60 mm for approx 15 mg of final conjugate)
with a degassed matrix suitable for the isolation of the conjugate from unconjugated
reagents. If the protein conjugated is an antibody, a matrix such as Bio-Gel A-0.5 m is
suitable. For other proteins, Sephadex G-200 or a similar column support may be appropriate,
depending on the size of the protein–avidin conjugate.
5. Collect 0.5–1-mL fractions. The first protein peak to elute contains the conjugate, however
the first or second fraction may contain some aggregates. Analyze each fraction absorbing
at 280 nm for biotin binding and assay it for the antibody or enzyme activity. High-performance
liquid chromatography (HPLC) may be also be performed for further purification,
if necessary.
3.2. Radioiodination Using IODO-BEADS
The radioiodination procedure (see Note 9) described here uses IODO-BEADS,
which contain the sodium salt of N-chlorobenzenesulfonamide immobilized on
nonporous, polystyrene beads. Immobilization of the oxidizing agent allows for easy
separation of the latter from the reaction mixture. This method also avoids the use of
reducing agents.