Cascade Blue 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.

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).

II. Covalent conjugation

Dissolve 5 mgs of Cascade Blue in 500 µl anhydrous DMSO immediately before use.

Add Cascade Blue to give a ratio of 600 µg per mg of antibody; mix immediately. (See notes in the detailed protocol about using different molar ratios of Cascade Blue to antibody).

Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.

Remove the unreacted Cascade Blue and exchange the antibody into "Storage Buffer" by gel filtration or dialysis. The unreacted dye will have the apparent color on the column; usually, the antibody conjugate will be too low a concentration to be colored: do not make the mistake of collecting the antibody by color visualization! (You can use a hand-held UV lamp in a darkened room to visualize the conjugate--it will appear faintly blue in comparison to the buffer).


"B Reaction Buffer": 100 mM carbonate, pH 8.4. For 1 liter: 8.4g 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.