Storing Reagents related to Antibody Conjugations

We keep all buffers at 4C.  Anhydrous DMSO is kept in a dessicator at room temperature; we designate a special bottle just for conjugations and use the highest-quality DMSO available.

Unconjugated antibodies are kept either at 4C or frozen. Any frozen protein material should always be centrifuged prior to use (for instance, in a microcentrifuge at 10,000g) to remove aggregates. Protein solutions should always be stored at relatively high concentrations (> 1 mg/ml).

Phycoerythrin and allophycocyanin are kept as saturated ammonium sulfate (SAS) precipitates at 4C. These are stable indefinitely as precipitates. To regenerate the protein solutions: centrifuge the precipitate at 10,000g at 4C for 10 min. Aspirate the supernatant; dissolve the precipitate in PBS. Dialyze against two changes of PBS (1000-fold volume excess) and one change of whatever buffer in which the phycobiliprotein is required.

Texas Red BSA is purchased from Molecular Probes as a solid; it is kept as a solid (in a dessicator at 4C) until use at which point it is dissolved in the buffer required for conjugation.

Reactive FITC, biotin, and Cascade Blue dyes are kept as solids at 4C, and are dissolved in anhydrous DMSO immediately prior to use. Cy5 and Cy7 reactive dyes are also kept as solids at 4C (dessicated) and dissolved immediately prior to use.

Final conjugates are kept at 4C in storage buffer; conjugates should never be frozen. If the concentration of the conjugate is less than 1 mg/ml protein, or if it is to be diluted for storage at a convenient titre, then it should be stored in a buffer containing protein. We typically dilute our conjugates in 0.1% gelatin (purchase a 2% gelatin solution from Sigma). We also use sodium azide (0.1%) to preserve all of our conjugates. We routinely stain with azide-preserved antibodies for sterile, viable sorting of cells—as long as cells are not allowed to warm up above room temperature, this is not a problem.