Conjugation of Cy5PE to Immunoglobulins:
Brief Protocol

See the detailed protocol for information.

I. Preparation of PE

Dialyze or exchange the PE into "C Reaction Buffer". Concentration before derivatization is typically 5-10 mg/ml. If the PE is stored as a SAS precipitate, it must be extensively dialyzed prior to use: 2 changes of 1 liter per ml PE of PBS before dialyzing against 1 liter per ml of "C Reaction Buffer".

Use 3.5 mg of R-PE per mg of IgG to be modified; this includes an extra 10% for loss during buffer exchanges.

To check the PE purity and concentration measure the absorbance at 280, 565 and 620 nm. (1 mg/ml of PE has an OD at 565nm of 8.2). A 565/620 ratio > 50 indicates adequate removal of contaminating phycocyanin; a 565/280 ratio > 5 indicates adequate removal of all other proteins.

II. Cy5 conjugation of PE

Dissolve the appropriate amount of bis-Cy5 in DMSO at 10 mg/ml. Add 49.8 nmol of Cy5 per mg of PE.

Incubate and rotate for 60 minutes. Exchange into "Dialysis Buffer" over gel filtration.

Take an absorbance spectrum of the product to ensure proper derivatization.

III. SMCC conjugation of Cy5PE

Prepare a 10 mg/ml stock solution of SMCC in dry DMSO immediately prior to use.

Add 11 µl of SMCC per mg of Cy5PE while vortexing. Wrap the reaction tube in aluminum foil and rotate at room temperature for 60 minutes.

Pass the derivatized Cy5PE over a gel filtration column pre-equilibrated with "Exchange Buffer".

IV. Reduction of 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.

V. Covalent conjugation

Add 3.2 mg of SMCC-Cy5PE 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").


"C Reaction Buffer": 500 mM carbonate, pH 9.0. For 1 liter: 17g Na2CO3, 28g NaHCO3

"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: 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