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98 Roque and Lowe
house using, for example, the FluoroTag FITC-conjugation kit (Sigma), with
which different conjugation ratios can be obtained (molar F/P of 2 is recommended
(13)). The conjugates may then be purified using pre-packed PD-10
columns (Amersham Biosciences-GE Healthcare) and characterized in terms of
the F/P ratio:
Molar F P = MW protein
389
×
A 495
/
195
A 280 − 035 × A 495 / 01%
280
where 01% is the absorption at 280 nm of a protein at 1 mg/ml; A 280 nm is the
absorbance measured at 280 nm.
b. Glass slides.
c. A fluorescence microscope with appropriate filters for the fluorophore used.
2. Affinity chromatography: When performing preparative small-scale assays,
disposable empty columns, for example, Bond Elut TCA® (4-ml propylene
columns with 20-μm frits) from Varian Inc. can be used. Alternatively, if choosing
an automatic system of sample/buffer loading and sample collection, for example,
the FPLC system from Amersham Biosciences-GE Healthcare, the affinity resins
must be properly packed in columns recommended by the supplier. The determination
of bound/washed and eluted protein can be performed with different
techniques, such as measurement of absorbance at 280 nm (using a conventional
spectrophotometer), quantitation of protein with colorimetric assays (such
as the Pierce BCA Protein Assay Reagent Kit from Pierce Biotechnology),
or quantitative ELISA (when utilizing small amounts of protein (14)), among
others.
2.3.2. Characterization of the Affinity Interactions Ligand-Protein
1. Partition equilibrium studies: Requires several Eppendorf tubes containing
solutions of the target proteins [usually 5–0.1 mg/ml in equilibration buffer] and
the agarose-immobilized ligand.
2. Competitive ELISA: Requires 96-well microtiter plates and ELISA plate reader
equipment. Proteins utilized were human IgG and human Fab (unconjugated
and conjugated to EZ-Link Activated Peroxidase (HRP) according to the
supplier instructions; Pierce Biotechnology) and PpL. Solutions needed included
coating buffer (0.05 M sodium carbonate-bicarbonate, pH 9.6); PBS-Tween
(PBST 20; 0.05% (v/v)), ligand 8/7 solution (82 μM in 50% DMF : PBS);
freshly prepared substrate solution (5 mM Na 2 HPO 4 , 2 mM citric acid, 1.85
mM o-phenylenediamine dihydrochloride (OPD; Merck) and 0.04% (v/v) H 2 O 2 );
stopping solution (50 μl of H 2 SO 4 , 2 M). Caution: OPD is harmful and considered
a potential carcinogenic; hydrogen peroxide and sulphuric acid are harmful and
corrosive—all these chemicals should be handled in a fume hood with safety
glasses and gloves.