View - ResearchGate

Rationally Designed Ligands for Use in Affinity Chromatography 103
3.3. Screening of Affinity Ligands and Characterization
of Affinity Interactions
3.3.1. Screening with the Conjugate FITC-Protein
Each synthesized affinity matrix (50 μl) is mixed with 100 μl of distilled
water in an Eppendorf tube, centrifuged for 2 min at 1430 × g, the supernatant
discarded and 2 × 100μl regeneration buffer added to the resin (see Fig. 4 ).
The components are gently mixed and centrifuged for 2 min at 1430 × g, the
supernatant discarded and 2 × 100μl of distilled water added to the resin. The
components are mixed and centrifuged for 2 min at 1430 × g, the supernatant
discarded and 2 × 100 μl of equilibration buffer added. The components are
again mixed and centrifuged for 2 min at 1430 × g. A conjugate FITC-target
protein (50 μl; 1 mg/ml in equilibration buffer) is added to the resin, and the
mixture incubated in the absence of light for 15 min with orbital agitation. After
this period, the resin is washed in the dark with 3×1mlequilibration buffer
(centrifuging the incubated resin with buffer at 1430 × g and then discarding the
supernatant). Each immobilized ligand matrix (1.5 μl) is placed on a microscope
slide and observed under a fluorescence microscope (FITC, exc = 495 nm;
em = 525 nm). The control experiments consist of repeating the procedure
described above using Sepharose® CL-6B, aminated agarose and control ligand
0/0. The results obtained by this screening system were compared with the data
resultant from the affinity chromatography test (13).
3.3.2. Screening of Affinity Ligands by Affinity Chromatography
(Performed at Room Temperature)
The affinity ligands (1 g of moist gel) are packed into 4-sml columns
(0.8 × 6 cm). Each matrix is washed with 2 × 3ml regeneration buffer and then
with distilled water to bring the pH to neutral. The resins are equilibrated with
10 ml of equilibration buffer. Protein to be tested is reconstituted to 1 mg/ml in
Fig. 4. Typical results obtained with the fluorescein isothiocyanate-based screening
system, showing examples of non-binding ligands (a), binding ligands (b) and strongly
binding ligands (c).