Ab | Antibody, about 2.5 mg/ml |
PE | R-Phycoerythrin, purified (Molecular Probes P-801 in 60% ammonium sulfate) |
PBS | Phosphate buffered saline without Ca2+/Mg2+ |
MeOH | Methanol |
DMSO | Dimethyl sulphoxide, anhydrous |
SPDP | 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (Sigma) |
SMCC | Succinimidyl trans-4-(N-maleimidylmethyl)cyclohexane-1-carboxylate (Molecular Probes) |
DTT | Dithiothreitol |
NEM | N-ethyl maleimide |
* | 10 ml Sephadex G-10 columns (2) |
Separation column | A long Sephacryl S-300 column. I used 117 x 1 cm. You can't really get away with anything smaller. Use a long thin one rather than a short fat one, since the latter did not work for me. Separation of the conjugate from the free reagents is tricky. |
* | Centricon 30 and Centriprep 30 centrifugal concentrators (Amicon) |
* | Room temperature is about 22 °C |