Cascade Blue conjugation of Annexin V:
Brief Protocol

For more information, see the detailed protocol.

I. Preparation of Annexin V

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.

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 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.