Protein Protocols Protein Protocols

358 Haugland and You
Because of its strength, the interaction between avidin and biotin cannot be used for
preparing matrices for affinity column purification, unless columns prepared with avidin
monomers are used (20). The biotin analog, iminobiotin, which has a lower affinity
for avidin, can be used for this purpose (21,22). Iminobiotin in reactive form is commercially
available, and the procedure for its conjugation is identical to that used for biotin.
Detailed, practical protocols for biotinylating antibodies at the lysine or at the carbohydrate
site, and a method to determine the degree of biotinylation are described in detail in this
chapter (see Notes 1–10 for review of factors that affect optimal conjugation and yield of
biotinylated antibodies).
2. Materials
2.1. Conjugation with Amine-Reactive Biotin
1. Reaction buffer: 1 M sodium bicarbonate, stable for about 2 wk when refrigerated. Dissolve
8.3 g of NaHCO3 in 100 mL of distilled water. The pH will be about 8.3. Dilute 1:10
before using to obtain a 0.1 M solution. Alternate reaction buffer: 0.1 M sodium phosphate,
pH 7.8. Dissolve 12.7 g Na2HPO4 and 1.43 g NaH2PO4 in 800 mL of distilled
water. Adjust pH to 7.8 if necessary. Bring the volume to 1000 mL. This buffer is stable
for 2 mo when refrigerated.
2. Anhydrous dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).
3. Phosphate-buffered saline (PBS): Dissolve 1.19 g of K2HPO4, 0.43 g of KH2PO4·H2O and 8.8 g NaCl in 800 mL of distilled water, adjust the pH to 7.2 if necessary or to the
desired pH, and bring the volume to 1000 mL with distilled water.
4. Disposable desalting columns or a gel-filtration column: Amicon GH-25 and Sephadex G-25
or the equivalent, equilibrated with PBS or buffer of choice.
5. Good-quality dialysis tubing as an alternative to the gel-filtration column when
derivatizing small quantities of antibody.
6. Biotin, biotin-X or biotin-XX succinimidyl ester: As with all succinimidyl esters, these
compounds should be stored well desiccated in the freezer.
2.2. Conjugation with Biotin Hydrazide at the Carbohydrate Site
1. Reaction buffer: 0.1 M acetate buffer, pH 6.0. Dilute 5.8 mL acetic acid in 800 mL distilled
water. Bring the pH to 6.0 with 5 M NaOH and the volume to 1000 mL. The buffer
is stable for several months when refrigerated.
2. 20 mM Sodium metaperiodate: Dissolve 43 mg of NaIO4 in 10 mL of reaction buffer,
protecting from light. Use fresh.
3. Biotin-X hydrazide or biotin-XX hydrazide.
4. DMSO.
5. Optional: 100 mM sodium cyanoborohydride, freshly prepared. Dissolve 6.3 mg of
NaBH3CN in 10 mL of 0.1 mM NaOH.
2.3. Determination of the Degree of Biotinylation
1. 10 mM 4'-Hydroxyazobenzene-2-carboxylic acid (HABA) in 10 mM NaOH.
2. 50 mM Sodium phosphate and 150 mM NaCl, pH 6.0. Dissolve 0.85 g of Na2HPO4 and
6.07 g of NaH2PO4 in 800 mL of distilled water. Add 88 g of NaCl. Bring the pH to 6.0 if
necessary and the volume to 1000 mL.
3. 0.5 mg/mL Avidin in 50 mM sodium phosphate and 150 mM NaCl, pH 6.0.
4. 0.25 mM Biotin in 50 mM sodium phosphate, and 150 mM NaCl, pH 6.0.