For more information, see the detailed protocol.
Note: it is
critical that sodium azide be completely removed from any Annexin V.
Dialyze or exchange over a column the Annexin V in "B Reaction
Buffer".
Measure the Annexin V concentration after buffer equilibration.
Dissolve 5 mgs of
Cascade Blue in 500 µl anhydrous DMSO immediately before use.
Add Cascade Blue to give a ratio of 50 µg per mg of Annexin V; mix immediately.
(See notes in the detailed protocol about using different molar rations of
Biotin to antibody).
Wrap the tube in foil; incubate and rotate at room temperature for 2 hours.
Remove the unreacted Cascade Blue 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.