For more information, see the detailed protocol.
Dissolve the TR-BSA
into "Dialysis Buffer", at a concentration of 10 mg/ml. Prepare 3 mg
TR-BSA per mg of antibody.
Prepare a 10 mg/ml stock solution of SMCC in dry DMSO immediately prior to use.
Add 45 µl of SMCC per mg of TR-BSA while vortexing. Wrap the reaction tube in aluminum foil and rotate at room temperature for 60 minutes.
Pass the SMCC-TR-BSA over a filtration column pre-equilibrated with "Exchange Buffer". Calculate the concentration of the SMCC-TR-BSA assuming a 90% recovery of starting material.
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 3 mg of TR-BSA
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