Protein Protocols Protein Protocols

352 Mao
terminus. The ε-amino group of lysine is moderately basic and reactive with acylating
reagents. The concentration of the free-base form of aliphatic amines below pH 8.0 is
very low. Thus, the kinetics of acylation reactions of amines by isothiocyanates,
succinimidyl esters, and other reagents is strongly pH-dependent. Although amine acylation
reactions should usually be carried out above pH 8.5, the acylation reagents
degrade in the presence of water, with the rate increasing as the pH increases. Therefore,
a pH of 8.5–9.5 is usually optimal for modifying lysines.
Where possible, the antibodies used for labeling should be pure. Affinity-purified,
fluorochrome-labeled antibodies demonstrate less background and nonspecific
fluorescence than fluorescent antiserum or immunoglobulin fractions. The labeling
procedures for the isothiocyanate derivatives of fluorescein and sulfonyl chloride
derivatives of rhodamine are given below (8). The major problem encountered is either
over- or undercoupling, but the level of conjugation can be determined by simple
absorbance readings.
2. Materials
1. IgG.
2. Borate buffered saline (BBS): 0.2 M boric acid, 160 mM NaCl, pH 8.0.
3. Fluorescein isothiocyanate (FITC) or Lissamine rhodamine B sulfonyl chloride (RBSC).
4. Sodium carbonate buffer: 1.0 M NaHCO3-Na2CO3 buffer, pH 9.5, prepared by titrating
1.0 M NaHCO3 with 1.0 M Na2CO3 until the pH reaches 9.5.
5. Absolute ethanol (200 proof) or anhydrous dimethylformamide (DMF).
6. Sephadex G-25 column.
7. Whatman DE-52 column.
8. 10 mM Sodium phosphate buffer, pH 8.0.
9. 0.02% Sodium azide.
10. UV spectrophotometer.
3. Methods
3.1. Coupling of Fluorochrome to IgG
1. Prior to coupling, prepare a gel-filtration column to separate the labeled antibody from the
free fluorochrome after the completion of the reaction. The size of the column should be
10 bed volumes/sample volume (see Note 1).
2. Equilibrate the column in phosphate buffer. Allow the column to run until the buffer level
drops just below the top of bed resin. Stop the flow of the column by using a valve at the
bottom of the column.
3. Prepare an IgG solution of at least 3 mg/mL in BBS, and add 0.2 vol of sodium carbonate
buffer to IgG solution to bring the pH to 9.0. If antibodies have been stored in sodium
azide, the azide must be removed prior to conjugation by extensive dialysis (see Note 2).
4. Prepare a fresh solution of fluorescein isothiocyanate at 5 mg/mL in ethanol or RBSC at
10 mg/mL in DMF immediately before use (see Note 3).
5. Add FITC at a 10-fold molar excess over IgG (about 25 µg of FITC/mg IgG). Mix well
and incubate at room temperature for 30 min with gentle shaking. Add RBSC at a 5-fold
molar excess over IgG (about 20 µg of RBSC/mg IgG), and incubate at 4°C for 1 h.
6. Carefully layer the reaction mixture on the top of the column. Open the valve to the column,
and allow the antibody solution to flow into the column until it just enters the bed resin. Carefully
add phosphate buffer to the top of the column. The conjugated antibody elutes in the
excluded volume (about one-third of the total bed volume).