For more information, see the detailed protocol.
Note: it is
critical that sodium azide be completely removed from any antibody: it will
react with the FITC and prevent conjugation.
Dialyze or exchange over a column the antibody in "Reaction Buffer".
Measure the antibody concentration after buffer equilibration. (For IgG, 1 mg/ml has an A(280) of 1.4).
Dissolve 10 mgs
(the entire contents of 1 vial; no need to weigh) of FITC in anhydrous DMSO
immediately before use.
Add FITC to give a ratio of 40-80 µg per mg of antibody; mix immediately. (See notes in the detailed protocol about using different molar rations of FITC to antibody).
Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.
Remove the unreacted FITC and exchange the antibody into "Storage Buffer" by gel filtration or dialysis.
Determine F/P and
protein concentration by measuring the absorbance at 280 and 495 nm.
IgG (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.4
IgM (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.2
IgG: 3.1 * A(495) / [A(280) - 0.31 * A(495) ]
IgM: 15.9 * A(495) / [A(280) - 0.31 * A(495) ]
Buffer": 500 mM carbonate, pH 9.5. For 1 liter: 17g Na2CO3, 28g NaHCO3
"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.