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 1.7 mg of APC per mg of IgG 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".
Prepare a fresh
solution of 1 M DTT (15.4 mg/100 µl) in distilled water.
IgG solutions should be at 4 mg/ml or higher for best results. Make each IgG solution 20 mM in DTT: add 20 µl of DTT stock per ml of IgG solution while mixing. Let stand at room temp for 30 minutes without additional mixing (to minimize reoxidation of cysteines to cystines).
Pass the reduced IgG 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 IgG. Carry out the conjugation as soon as possible after this step.
Add 1.5 mg of
SMCC-APC per mg of IgG. 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 34 µg (3.4 µl) per mg of IgG. 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