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Fig. 1. Schematic of cationic nylon filter binding assay for growth factor/heparan sulfate proteoglycan interactions. Filtration of various mixtures of growth factor, heparan sulfate proteoglycan (E-HS), and compounds that block either the heparin-binding domain on the growth factor or the protein-binding domain on the heparan sulfate chains, through cationic nylon filters can be used to measure the various binding affinities quantitatively. (1) Cationic nylon filters do not specifically retain small proteins such as FGF-2. (2) FGF-2 bound to heparan sulfate proteoglycans will be retained on the filter, providing a direct measure of FGF-2/heparan sulfate proteoglycan binding. (3) Compounds that bind to E-HS and block the growth factor-binding domain can reduce FGF-2 retention on the filter in relation to the binding affinity of the inhibitor compound for E-HS. (4) Compounds that bind to FGF-2 and block its interaction with E-HS can reduced FGF-2 retention in relation to the binding affinity of the inhibitor compound for FGF-2. As an example we have provided a description of how this assay can be used to determine the FGF-2-binding affinity for E-HS and sucrose octasulfate, and the binding affinity of E-HS for protamine sulfate.

3. Method

3.1. Experimental Protocol

1. Label and arrange 0.5-mL microcentrifuge tubes—1 per sample, 3 per condition.

2. Prepare incubation buffer—Buffer may be made in advance and filter-sterilized for later use. No degassing is needed.

3. Add the needed incubation buffer to each vial—the volume should be 0.2 mL minus the volume of 125I-bFGF, inhibitor, and E-HS needed. Controls should be conducted with vials containing only 125I-bFGF, and 125I-bFGF and the inhibitors, to determine the non-proteoglycan-mediated adsorption of 125I-bFGF to the filter. These values should be substrated from the experimental points with E-HS to determine the level of 125I-bFGF bound to E-HS.

4. Add the E-HS, inhibitor, and 125I-bFGF in that order. As a standard assay we have used 40 ng of E-HS and 0.2 ng of 125I-bFGF in a final volume of 0.2 mL. Under these conditions greater than 80% of the E-HS is retained with and without bFGF addition, and only ~5% of the bFGF will adsorb to the filter nonspecifically. Initial experiments should be conducted with a range of 125I-bFGF concentrations to determine the binding affinity and capacity of each E-HS preparation before inhibitor competition data can be analyzed. After each addition of 125I-bFGF, vortex gently to mix and place in an incubation rack. If the assay is done at a temperature other than room temperature (25°C), be sure that the buffers are preequilibrated at the desired temperature and that vials are placed immediately in a temperature equilibrated rack.

5. Incubate for desired time (generally 60 min to reach a steady state in bound versus unbound bFGF).

6. If incubation is shorter then 30 min, the cationic membrane should be prepared prior to incubation (steps 3-5). The membrane is cut to fit the apparatus (cover all holes) and then incubated on a rocking platform for at least 20 min in incubation buffer. Longer incubations (up to 1 h) do not adversely affect the assay.

7. The membrane is placed on the dot-blot apparatus and the top plate is tightened using a criss-cross pattern. The apparatus is attached to a vacuum pump (GAST, model ROA-P131-AA, Manufacturing Corp., Benton Harbor, MI) and the apparatus tightened again under vacuum. Experiments should be done under a 25 mmHg vacuum.

8. Close off the apparatus to the vacuum and, using a multipipeter, add 0.2 mL of incubation buffer to each well.

9. When the incubation is within 30 s of completion, turn on the vacuum and pull the wash buffer through the membrane. Add samples (0.2 mL/well) in sets of three. Wash each well of the triplicate set once with 0.2 mL of incubation buffer before adding the next set of samples to the membrane. Allow all sample fluid to filter before adding the incubation buffer. Repeat for each set of triplicate samples. Air bubbles may arise and interfere with the filtration. Gently pipet the liquid near the surface of the membrane to mix, being careful not to puncture the membrane.

10. After all samples have been added, do two additional washes with 0.2 mL of incubation buffer per well.

11. Loosen the screws holding the unit in place and remove the top plate. Turn off the vacuum and carefully remove the membrane.

12. Allow the membrane to dry sample side up on a paper towel and then cut out the individual membrane dots corresponding to each individual well. Place each sample dot in a vial and count the radioactive bFGF in a gamma counter. Sample data are shown in Fig. 2.

Fig. 2. Inhibitors of bFGF-E-HS Binding. E-HS (200 ng/mL), bFGF (0.056 nM), and inhibitor ([A] sucrose octasulfate or SOS [B] protamine sulfate or PS) were incubated for 1 h and then filtered across the cationic membrane. bFGF retention following incubation with SOS or PS in the absence of E-HS was subtracted from total binding as nonspecific binding. 100% bFGF bound corresponds to specific bFGF retention following incubation with E-HS in the absence of inhibitor. The average ± standard error of at least triplicate samples is shown.

Fig. 2. Inhibitors of bFGF-E-HS Binding. E-HS (200 ng/mL), bFGF (0.056 nM), and inhibitor ([A] sucrose octasulfate or SOS [B] protamine sulfate or PS) were incubated for 1 h and then filtered across the cationic membrane. bFGF retention following incubation with SOS or PS in the absence of E-HS was subtracted from total binding as nonspecific binding. 100% bFGF bound corresponds to specific bFGF retention following incubation with E-HS in the absence of inhibitor. The average ± standard error of at least triplicate samples is shown.

3.2 Analysis

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