Texas Red-BSA conjugation of Antibodies:
Brief Protocol
For more information, see the detailed protocol.
I. Derivatization of the TR-BSA
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.
II. Reduction of the IgG
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.
III. Covalent conjugation
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").
Buffers
"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:
10.90 g MES, 0.74 gm EDTA
"Storage Buffer": 10 mM Tris, 150 mM NaCl, pHix, pH 8.2. For 1
liter: 1.42g TRIZMA 8.0, 8.77g NaCl, 3-4 drops pHix
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CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu