See the detailed protocol for information.
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.
Dissolve the
appropriate amount of bis-Cy7 in DMSO at 10 mg/ml. Add 83.3 nmol of Cy7 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.
Prepare a 10 mg/ml
stock solution of SMCC in dry DMSO immediately prior to use.
Add 11 µl of SMCC per mg of Cy7PE while vortexing. Wrap the reaction tube in
aluminum foil and rotate at room temperature for 60 minutes.
Pass the derivatized Cy7PE over a gel filtration column pre-equilibrated with
"Exchange Buffer".
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.
Add 3.2 mg of
SMCC-Cy7PE 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