For more information, see the detailed protocol.
Note: it is
critical that sodium azide be completely removed from any antibody.
Dialyze or exchange over a column the antibody in "B Reaction Buffer".
Measure the antibody concentration after buffer equilibration. (For IgG, 1 mg/ml has an A(280) of 1.4).
Dissolve 10 mgs of
Biotin in 1 ml anhydrous DMSO immediately before use.
Add Biotin to give a ratio of 80 µg per mg of antibody; mix immediately. (See notes in the detailed protocol about using different molar rations of Biotin to antibody).
Wrap the tube in foil; incubate and rotate at room temperature for 2 hours.
Remove the unreacted Biotin and exchange the antibody into "Storage Buffer" by gel filtration or dialysis.
"B Reaction Buffer": 100 mM carbonate, pH 8.4. For 1 liter: 8.4g
"Storage Buffer": 10 mM Tris, 150 mM NaCl, 0.1% (w/v) NaN3, pH 8.2. For 1 liter: 1.42g TRIZMA 8.0, 8.77g NaCl, 5 ml 20% NaN3.