Protein Protocols Protein Protocols

360 Haugland and You
5. Dissolve 10 mg of the biotin hydrazide of choice in 0.25 mL of DMSO to obtain a 40 mg/mL
solution, warming if needed. This will yield a 107 mM solution of biotin-X hydrazide or
an 80 mM solution of biotin-XX hydrazide. These solutions are stable for a few weeks.
6. Calculate the amount of biotin hydrazide solution needed to obtain a final concentration
of approx 5 mM, and add it to the oxidized antibody. When using biotin-X hydrazide, 1 vol
of hydrazide should be added to 20 vol of antibody solution. When using biotin-XX hydrazide,
1 vol of hydrazide should be added to 15 vol of antibody solution.
7. Incubate for 2 h at room temperature with gentle stirring.
8. This step is optional. The biotin hydrazone–antibody conjugate formed in this reaction
(steps 6 and 7) is considered by some researchers to be relatively unstable. To reduce the
conjugate to a more stable, substituted hydrazide, treat the conjugate with sodium
cyanoborohydride at a final concentration of 5 mM by adding a 1/20 vol of a 100-mM
stock solution. Incubate for 2 h at 4°C (see Note 5).
9. Purify the conjugate by any of the methods described for biotinylating antibodies at the
amine site (see Subheading 3.1., step 6).
3.3. Determination of the Degree of Biotinylation
The dye HABA interacts with avidin yielding a complex with an absorption maximum
at 500 nm. Biotin, because of its higher affinity, displaces HABA, causing a decrease in
absorbance at 500 nm proportional to the amount of biotin present in the assay.
1. To prepare a standard curve, add 0.25 mL of HABA reagent to 10 mL of avidin solution.
Incubate 10 min at room temperature and record the absorbance at 500 nm of 1 mL avidin–HABA
complex with 0.1 mL buffer, pH 6.0. Distribute 1 mL of the avidin–HABA
complex into six test tubes. Add to each the biotin solution in a range of 0.005–0.10 mL.
Bring the final volume to 1.10 mL with pH 6.0 buffer, and record the absorbance at
500 nm of each concentration point. Plot a standard curve with the nanomoles of biotin vs
the decrease in absorbance at 500 nm. An example of a standard curve is illustrated in Fig. 3.
2. To measure the degree of biotinylation of the sample, add an aliquot of biotinylated
antibody of known concentration to 1 mL of avidin–HABA complex. For example, add
0.05–0.1 mL of biotinylated antibody at 1 mg/mL to 1 mL of avidin–HABA mixture. Bring
the volume to 1.10 mL, if necessary, incubate for 10 min, and measure the decrease in
absorbance at 500 nm.
3. Deduct from the standard curve the nanomoles of biotin corresponding to the observed
change in absorbance. The ratio between nanomoles of biotin and nanomoles of antibody
used to displace HABA represents the degree of biotinylation, as seen from the following
equation:
[(nmol biotin × 145,000 × 10 –6 )/(mg/mL antibody × 0.1 mL)]
= (mol of biotin/mol of antibody) (3)
where 145,000 represents the mol wt of the antibody and 0.1 mL is the volume of 1 mg/mL
of biotinylated antibody sample.
4. Notes
4.1. Factors that Influence the Biotinylation Reaction
1. Protein concentration: As in any chemical reaction, the concentration of the reagents is a
major factor in determining the rate and the efficiency of the coupling. Antibodies at a
concentration of 5–20 mg/mL will give better results; however, it is often difficult to have
such concentrations or even such quantities available for conjugation. Nevertheless, the