For more information, see the detailed protocol.
Dialyze or exchange
the APC into "Dialysis Buffer". Concentration before derivatization
is typically 5-10 mg/ml. If the APC is stored as a SAS precipitate, it must be
extensively dialyzed prior to use: 2 changes of 1 liter per ml APC of PBS
before dialyzing against 1 liter per ml of "Dialysis Buffer".
Use 3-6 mg of APC per mg of Annexin V to be modified; this includes an extra
10% for loss during buffer exchanges.
To check the APC purity, measure the absorbance at 280, 620 and 655 nm. (1
mg/ml of APC has an OD at 655nm of 5.9). A 655/620 ratio >1.4 indicates
adequate removal of PC; a 655/280 ratio > 4 indicates adequate removal of
all other proteins.
Prepare a 10 mg/ml
stock solution of SMCC in dry DMSO immediately prior to use.
Add 6 µl of SMCC per mg of APC while vortexing. Wrap the reaction tube in
aluminum foil and rotate at room temperature for 60 minutes.
Pass the derivatized APC over a gel filtration column pre-equilibrated with
"Exchange Buffer".
Dissolve (or
dialyze or exchange) the Annexin V into any standard PBS solution
(azide-free!).
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 3-6 mg of
SMCC-APC 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").
"Dialysis
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