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Phycoerythrin conjugation protocol

David's method modified from references (2) and (3). I used this method to conjugate a mouse IgG2a monoclonal antibody (IB4). It works well.

Reagents

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

Method

1. Preparation of PE: R-PE from Molecular Probes. Part number P-801, R-PE in 60% NH₄SO₄ at 4 mg/ml.
0.25 ml of P-801. Spin 10 mins on low in microfuge. Discard supernatant. Resuspend PE in 0.25 ml PBS. Dialyze twice for 30 mins versus 150 ml PBS at room temperature.
2. SPDP modification of PE: Adjust PE to approx 1.4 mg/ml (=0.7 ml) in PBS
Add 16 µl SPDP (1.3 mg/ml in MeOH)
Incubate at room temperature for 2.5 hours
*** You must time steps 3 and 4 to finish at the same time, since the reaction products are unstable ***
3. SMCC modification of antibody: 1 ml purified antibody at approx 2.5 mg/ml in PBS. Antibody must be fairly pure - i.e. purify on protein A or G column before conjugating.
Add 20 µl SMCC, 1.7 mg/ml in DMSO.
Incubate for 1 hour.
4. DTT treatment of SPDP-PE: Take product from step (2) and add 30 µl DTT (0.5M, i.e. 77 mg/ml in PBS).
Incubate for 30 mins at room temperature.
5. Purification of reactants: Separate derivatized PE and Ab from activators on G-10 columns. You should now have approximately 2 ml of each reactant.
6. Conjugation: Mix derivatized reagents and incubate at 4 °C overnight on rotary mixer.
7. Stop reaction: Add 80 µl N-ethyl maleimide (0.1 mM) to reaction mixture to stop reaction. Agitate and incubate 30 mins at room temperature
8. Concentrate product: Concentrate to 0.5 ml or less on Centricon concentrator (Centricon 30 or 100 are fine)
9. Separate conjugate: Apply to top of separation column. Elute with PBS. Pump column at approximately 0.3 ml/min.
Order of elution is conjugate first (MW ~400,000) then free PE (MW ~240,000) then free Ab (MW ~150,000). Begin collecting 1.0 ml fractions when pink band approaches bottom of column and continue for 10 fractions after it has disappeared.
10. Evaluate separation: Measure OD₂₈₀ and OD₅₆₅ - the former is protein, the latter the PE absorption peak. Test activity of each fraction against known reactive cells by flow cytometry. Plot all these results on a graph and estimate where the most conjugate is (See figure below). Pool these fractions.
11. Final concentration: Concentrate pooled conjugate fractions using Centriprep and Centricon concentrators.
Add 0.5% sodium azide. Store at 4 °C protected from light. Do not freeze.

Figure 1. Analysis of column eluate fractions from PE conjugation of monoclonal antibody IB4 (murine IgG2a, anti-human CD18). The optical density of each fraction was measured at 280 nm and 565 nm (OD₂₈₀ and OD₅₆₅ respectively). An aliquot of each fraction was used to stain human neutrophils which were then analyzed using a FACScan flow cytometer (Becton-Dickinson). Median cellular fluorescence intensity in the FL2 (orange fluorescence) channel is shown.

References

Chambers JD, Simon SI, Berger EM, Sklar LA, Arfors K-E. Endocytosis of beta₂ integrins by stimulated human neutrophils analyzed by flow cytometry. Journal of Leukocyte Biology 53: 462-469 (1993)
Kronick MN, Grossman PD. Immunoassay techniques with fluorescent phycobiliprotein conjugates. Clinical chemistry 29: 1582-1586 (1983)
Oi VT, Glazer AN, Stryer L. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. Journal of Cell Biology 93: 981-986 (1982)

David Chambers: 960614; 960621