Protein Protocols Protein Protocols

368 Haugland and Bhalgat
(23), with minimal generation of aggregates. A similar protocol is described here for
conjugation of 5 mg of avidin to antibodies or enzymes. Modifications of the procedure
for conjugation of avidin to thiolated oligonucleotides and peptides are described
in Notes 2 and 5, respectively. Although the protocol described in this section uses
avidin for conjugation, it can be applied for the preparation of conjugates using either
avidin, streptavidin, deglycosylated avidin, or NeutrAvidin avidin.
1. Dissolve 5 mg of avidin (76 nanomol) in 0.5 mL of 0.1 M phosphate buffer to obtain a
concentration of 10 mg/mL.
2. Weigh 3 mg of SPDP and dissolve it in 0.3 mL of DMSO to obtain a 10 mg/mL solution.
This solution must be prepared fresh immediately before using. Vortex-mix or sonicate to
ensure that the reagent is completely dissolved.
3. Slowly add 12 µL (380 nanomoles) of the SPDP solution (see Note 3) to the stirred solution
of avidin. Stir for 1 h at room temperature.
4. Purify the thiolated avidin on a 7 × 250 mm size exclusion column, such as Sephadex G-25
equilibrated in 0.1 M phosphate buffer.
5. Determine the degree of thiolation (optional):
a. Prepare a 100 mM solution of DTT by dissolving 7.7 mg of the reagent in 0.5 mL of
distilled water.
b. Transfer the equivalent of 0.3–0.4 mg of thiolated avidin (absorbance at 280 nm of a
1.0 mg/mL avidin solution = 1.54) and dilute to 1.0 mL using 0.1M phosphate buffer.
Record the absorbance at 280 nm and at 343 nm.
c. Add 50 mL of DTT solution. Mix well, incubate for 3–5 min at room temperature and
record the absorbance at 343 nm.
d. Using the extinction coefficient at 343 nm of 8.08 × 10 3 /cm/M (24), calculate the
amount of pyridine-2-thione liberated during the reduction, which is equivalent to
the number of thiols introduced on avidin, using the following equation along with the
appropriate extinction coefficient shown in Table 1:
Number of thiols/avidin = [∆A 343/(8.08 × 10 3 )] × (A 280–0.63∆A 343)] (1)
where ∆Α343 = change in absorbance at 343 nm; EM avidin = molar extinction coefficient;
and 0.63∆Α343 = correction for the absorbance of pyridyldithiopropionate at 280 nm (24).
6. Equation 1 allows the determination of the average number of moles of enzyme or
antibody that can be conjugated with each mole of avidin (see Note 6). For a 1:1 proteinavidin
conjugate, avidin should be modified with 1.2–1.5 thiols/mol. Thiolated avidin
prepared by the above procedure can be stored in the presence of 2 mM sodium azide at
4°C for 4–6 wk.
3.1.2. Maleimide Derivatization of the Antibody or Enzyme
In this step, which should be completed prior to the deprotection of thiolated avidin,
some of the amino groups from the antibody or enzyme are transformed into maleimide
groups by reacting with the bifunctional crosslinker, SMCC. (see Note 7).
1. Dissolve or, if already in solution, dialyze the protein in 0.1 M phosphate buffer to obtain
a concentration of 2–10 mg/mL. If the protein is an antibody, 11 mg are required to obtain
an amount equimolar to 5 mg of avidin (see Note 6).
2. Prepare a fresh solution of SMCC by dissolving 5 mg in 0.5 mL of dry DMSO to obtain a
10 mg/mL solution. Vorte-mix or sonicate to assure that the reagent is completely dissolved.
3. While stirring, add an appropriate amount of SMCC solution to the protein solution to
obtain a molar ratio of SMCC to protein of approx 10. (If 11 mg of an antibody is the
protein used, 30 µL of SMCC solution is required.)