Fluorescein (mistakenly abbreviated by its commonly-used reactive
isothiocyanate form, FITC) is currently the most commonly-used fluorescent dye
for FACS analysis. FITC is a small organic molecule, and is typically
conjugated to proteins via primary amines (i.e., lysines). Usually, between 3
and 6 FITC molecules are conjugated to each antibody; higher conjugations can result
in solubility problems as well as internal quenching (and reduced brightness).
Thus, an antibody will usually be conjugated in several parallel reactions to
different amounts of FITC, and the resulting reagents will be compared for
brightness (and background stickiness) to choose the optimal conjugation ratio.
Fluorescein is typically excited by the 488 nm line of an argon laser, and
emission is collected at 530 nm. (See the fluorescence
Refer to notes about the following procedures used by this protocol:
You can also use the short, less-detailed protocol for reference.
I. Preparation of antibody
II. Covalent conjugation
III. Characterizing the conjugate
The entire conjugation can be performed in about a half-day. In addition to
the materials listed below, you will need to have a solution of your antibody
at a concentration (optimally) of at least 2 mg/ml. The extent of FITC conjugation
to the antibody may depend on the concentration of antibody in solution; for
consistent conjugations, use a consistent concentration. You should be familiar
with how to use a desalting column and how to take absorbance spectra.
The reactive fluorescein molecule, fluorescein isothiocyanate, is unstable. Once a vial has been cracked and the FITC solubilized, it should be used immediately. Since single vials of FITC contain sufficient material for ~100 mgs of antibody, it is economical to perform multiple FITC conjugations on the same day.
When first conjugating an antibody, a range of FITC to antibody concentrations should be compared. The protocol suggests 40-80 µg per mg of antibody; for a first-time titration of FITC, try a range of 10 to 400 µg FITC per mg of antibody (for instance, 10, 40, 80, 160, 320 µg per mg). Compare each conjugate by staining (you should perform a titration of antibody on cells for each reagent to determine the optimal staining concentration). Select the conjugate with the brightest "positive" cells which still has low background on "negative" cells.
Note: it is critical that sodium azide be completely removed from any
antibody: it will react with the FITC and prevent conjugation.
Dialyze or exchange over a column the antibody in "Reaction Buffer". Note that the BioRad protein reagent kit reacts spontaneously in "Reaction Buffer"; it is difficult (but not impossible) to determine which column fractions contain the protein by this method... use of a spectrophotometer is preferred. See hints on column separations of nonfluorescent proteins.
Measure the antibody concentration after buffer equilibration. (For IgG, 1 mg/ml has an A(280) of 1.4). If the antibody concentration is less than 1 mg/ml, the conjugation will probably be sub-optimal. If necessary, dilute the antibody to a concentration of 4 mg/ml.
FITC is covalently coupled to primary amines (lysines) of the
Dissolve 10 mgs (the entire contents of 1 vial; no need to weigh) of FITC in 1 mL anhydrous DMSO immediately before use.
Add FITC to give a ratio of 40-80 µg per mg of antibody; mix immediately. (See notes above about using different molar rations of FITC to antibody).
Wrap the tube in foil; incubate and rotate at room temperature for 1 hour.
Remove the unreacted FITC and exchange the antibody into "Storage Buffer" by gel filtration or dialysis.
Determine F/P and protein concentration by
measuring the absorbance at 280 and 495 nm.
IgG: 1 mg/ml has an A(280) of 1.4; mw = 150,000
IgM: 1 mg/ml has an A(280) of 1.2; mw = 900,000
Fluorescein: 1 mM has an A(495) of 68 and an A(280) of 11.8.
F/P values of 3-10 are probably optimal for any partciular IgG.
IgG (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.4
IgM (mg/ml) = [ A(280) - 0.31 * A(495) ] / 1.2
IgG: 3.1 * A(495) / [A(280) - 0.31 * A(495) ]
IgM: 15.9 * A(495) / [A(280) - 0.31 * A(495) ]
For column separations, we often use one of two types of pre-poured columns:
For 1.25ml to 2.5ml sample volumes: PD-10 (Sephadex G-25M), Amersham, catalog No. 17-0851-01.
For <0.5 ml sample volumes: NAP5 columns (Sephadex G-25 DNA grade), Amersham, catalog No 17-0853-02.
FITC - fluorescein isothiocyanate
Molecular Probes, catalog No. F1906
DMSO - anyhydrous dimethyl sulfoxide
Aldrich, catalog No. 27,685-5.
Note: keep the DMSO absolutely dry at all times. We keep the bottle in a dessicator. Pour out an amount of DMSO sufficient for your need and then pipette that; don't pipetter directly into the bottle.
NaHCO3 - sodium bicarbonate
J. T. Baker, catalog No. 3508-05, mw 84.01
NaCO3 - sodium carbonate
J. T. Baker, catalog No. 3602-01, mw 106
NaCl - Sodium Chloride
Sigma, Catalog No S-3014, mw 58.44
TRIZMA pre-Set crystals 8.0 - Combination of Tris base and TrisHCl
Sigma, catalog No. T4753, average mw 141.8
NaN3 – Sodium Azide
Sigma, catalog No S-2002, mw 65
500 mM carbonate, pH 9.5
To make 1 Liter:
Note: sodium azide cannot be added to this buffer
10 mM Tris, 150 mM NaCl, 0.1% NaN3, pH 8.2
To make 1 Liter:
1.42g TRIZMA 8.0
pH to 8.2
See hints on storing buffers.
This protocol is based on an original protocol devised by Aaron Kantor.