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 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.