FITC conjugation of Antibodies:
Brief Protocol

For more information, see the detailed protocol.

I. Preparation of antibody

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).

II. Covalent conjugation

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.

III. Characterizing the conjugate

Determine F/P and protein concentration by measuring the absorbance at 280 and 495 nm.

Protein concentration:
IgG (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.4
IgM (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.2

F/P ratio:
IgG: 3.1 * A(495) / [A(280) - 0.31 * A(495) ]
IgM: 15.9 * A(495) / [A(280) - 0.31 * A(495) ]


"Reaction 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.