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104 Roque and Lowe
equilibration buffer and the absorbance at 280 nm measured. Protein solution
(1 ml) is loaded onto each column. The columns are washed with equilibration
buffer until the absorbance of the samples at 280 nm reaches ≤0.005. Bound
protein is eluted with the elution buffer (1 ml fractions collected). After elution,
the columns are regenerated with regeneration buffer, followed by distilled
water and equilibration buffer, and stored at 0–4°C in 20%(v/v) ethanol.
3.3.3. Characterization of Affinity Interactions by Partition
Equilibrium Experiments
The immobilized ligand in study is treated with regeneration buffer and then
equilibrated in equilibration buffer. A series of Eppendorf tubes are prepared
with 1 ml of standard protein solutions in equilibration buffer (5 tubes at –0.1
mg/ml; confirm concentration by A 280 nm measurement). Immobilized ligand
(0.1 g of moist weight gel previously dried under vacuum in a sintered funnel) is
added to each Eppendorf tube and incubated for 24 h, at room temperature and
under orbital agitation. After this period, the Eppendorf tubes are centrifuged
(1 min; 1430 g) to settle the matrix, and the supernatant is taken to measure the
A 280 nm . The control experiment comprised of incubating the partitioning solute
with unmodified Sepharose® CL-6B. The data collected from these experiments
are then utilized to calculate the affinity constants for the interaction of the
ligand with the target protein (see Note 6).
3.3.4. Competitive ELISA (15)
The wells of an ELISA (see Fig. 5) microplate were coated with 100 μl
of PpL (10 μg/ml) in coating buffer overnight at 0–4°C. After three washing
steps with PBST, the plate was blocked with PBST (200 μl/well) and incubated
for1hatroom temperature. The plate was extensively washed with PBST and
100 μl of PBST added to each well except the first row. For the determination
of the inhibition of ligand 8/7 in the interaction between PpL with IgG and Fab,
200 μl of ligand 8/7 solution was added to the first row and diluted (1:2) by
transferring 100 μl from well to well along the plate. Protein conjugated to HRP
(hIgG-HRP, 1:1,000; hFab-HRP, 1:500 in PBST) (100 μl) was added to all wells
and the plate incubated for2hatroom temperature. After incubation, the plates
were carefully and extensively washed with PBST. Substrate solution (100 μl)
was added to the wells. The plates were incubated at room temperature in the
dark (10 min: hIgG-HRP; 30 min: hFab-HRP). After the incubation period, 50
μl of stopping solution was added to each well and the absorbance read at 490
nm. The control wells contained (i) no protein-HRP, (ii) no protein-HRP and