For more information, see the detailed protocol.
Dialyze or exchange the PE into "Dialysis Buffer". Concentration
before derivatization is typically 5-10 mg/ml. If the PE is stored as a SAS
precipitate, it must be extensively dialyzed prior to use: 2 changes of 1 liter
per ml PE of PBS before dialyzing against 1 liter per ml of "Dialysis
Use 6-12 mg of R-PE per mg of Annexin V to be modified.
To check the PE purity and concentration measure the absorbance at 280, 565 and 620 nm. (1 mg/ml of PE has an OD at 565nm of 8.2). A 565/620 ratio > 50 indicates adequate removal of contaminating phycocyanin; a 565/280 ratio > 5 indicates adequate removal of all other proteins.
Prepare a 10 mg/ml stock solution of SMCC in dry DMSO immediately prior to
Add 11 µl of SMCC per mg of PE while vortexing. Wrap the reaction tube in aluminum foil and rotate at room temperature for 60 minutes.
Pass the derivatized PE over a gel filtration column pre-equilibrated with "Exchange Buffer".
Dissolve (or dialyze or exchange) the Annexin V into any standard PBS
Dissolve 2-IT in the same PBS solution at a concentration of 10 mg/ml. Use immediately: for a molar ratio of 4, add 15.2 µg of 2-IT per mg of Annexin. See notes in the detailed protocol about using different molar ratios of 2-IT to Annexin V.
Incubate and rotate at room temperature for 60 minutes.
Prepare a fresh solution of 1 M DTT (15.4 mg/100 µl) in distilled water.
Annexin V solutions should be at 4 mg/ml or higher for best results. Make each Annexin V solution 20 mM in DTT: add 20 µl of DTT stock per ml of Annexin V solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced Annexin V over a filtration column pre-equilibrated with "Exchange Buffer". Collect 0.25 ml fractions off the column; determine the protein concentrations and pool the fractions with the majority of the Annexin V. Carry out the conjugation as soon as possible after this step.
Add 6-12 mg of SMCC-PE per mg of Annexin V. Wrap the reaction tube in
aluminum foil and rotate for 60 minutes at room temp.
Prepare a fresh solution of 10 mg NEM in 1.0 ml dry DMSO.
Add 20 µg (2.0 µl) per mg of Annexin V. Wrap and rotate for 20 minutes at room temperature.
The product can be either dialyzed or exchanged over a column into an appropriate buffer (e.g. "Storage Buffer").
Buffer": 50 mM Sodium phosphate, 1 mM EDTA, pH 7.0. For 1 liter: 13.41g
Sodium phosphate dibasic (7*H2O); 0.37 g EDTA
"Exchange Buffer": 50 mM MES, 2 mM EDTA, pH. 6.0. For 1 liter: 9.76 g MES, 0.74 gm EDTA
"Storage Buffer": 10 mM Tris, 150 mM NaCl, 0.1% (w/v) NaN3 (azide), pH 8.2. For 1 liter: 1.42g TRIZMA 8.0, 8.77g NaCl, 5 mL 20% NaN3
Standard PBS: must be free of sodium azide