For more information, see the detailed protocol.
Note: it is
critical that sodium azide be completely removed from any Annexin V: it will
react with the FITC and prevent conjugation.
Dialyze or exchange over a column the Annexin V in "Reaction Buffer".
Measure the Annexin V concentration after buffer equilibration.
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 10-20 µg per mg of Annexin V; mix immediately. (See
notes in the detailed protocol above about using different molar rations of
FITC to Annexin V).
Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.
Remove the unreacted FITC and exchange the Annexin V 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.