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 Cy5 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 the Cy5 in anhydrous DMSO immediately before use, at a
concentration of 10 mg/ml. Do not delay between weighing out the Cy5 and
dissolving it in DMSO; likewise, do not delay the addition of the solubilized
material to the antibody.
For the optimal ratio of 5:1, add 40 µg Cy5 per mg of antibody; mix
immediately.
Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.
Remove the unreacted Cy5 and exchange the antibody into "Storage
Buffer" by gel filtration or dialysis.
"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.