Methods for Preparing Extracellular Matrix and Quantifying Insulin-like Growth Factor-Binding Protein Binding to the ECM - Methods in Molecular Biology: Extracellular Matrix Protocols - page 215

the bead is removed and the mixture purified by Sephadex G-100 chromatogra-
phy. Add about 80,000 mL 125 I-IGFBP-5 to the solutions containing each con-
centration of unlabeled IGFBP-5 in a final incubation volume of 2.0 mL.

7. Incubate the membranes with the appropriate treatments overnight on a shaker at

4 C. Wash the membranes three times for 10 min each with 30 m M sodium phos-

phate containing 0.2% Triton X-100, pH 7.4, at 4

°

° C. Allow membranes to dry.

8. Cut the two slot sections from the membranes and place each 2 slot section in

12 75 mm polystyrene tube. Count the bound 125 I in a gamma spectrometer.

Plot the results to determine competition. Nonspecific binding is determined by
subtracting the counts per minute bound in the presence of 50

×

g/mL unlabeled
IGFBP-5. This value should be <15% of the total binding and should be sub-
tracted from each data point.

µ

3.3. Procedures to Verify that the ECM has been Extracted
Save the primary urea extract and secondary Laemmli sample buffer extract
and test them in the following manner.
1. Western ligand blotting of IGFBP-5: Concentrate 0.5 mL of sample on a

Millipore Ultrafree 0.5-centrifuge filter Biomax 10k NMWL membrane to
40 L sample to 10 mL of 100% glycerol, and then add to a

12.5% SDS polyacrylamide gel. After the separation, transfer the proteins to an
Immobilon membrane. Incubate the filter for 14 h with 125 I-IGF-I (specific activ-
ity 125

µ L. Add the 40- µ

g). Add 500,000 cpm to 4 mL of blotting buffer ( see ref. (10) for the
buffer composition). The filter is washed three times, and the bound 125 I-IGF-I visu-
alized by autoradiography. A band with an Mr estimate of 30-32 kDa should be
seen. That this is IGFBP-5 can be confirmed by immunoblotting (11) .

µ Ci/ µ

2. To test the ECM extract binding to IGFBP-5, using a slot blotter apparatus, con-

centrate 170 L of the ECM extract on an Immobilon filter, as described previ-

ously. Unlabeled IGFBP-5 should compete with 125 I-IGFBP-5 for binding (e.g.,
>50% reduction in binding) when 500 ng/mL is added. The 125 I-IGFBP-5 bind-
ing assay protocol is outlined above.

µ

3. Immunoblot the concentrated ECM for vitronectin ( see Note 3 ). pSMC ECM

contains a high concentration of vitronectin. Concentrate 170 L of ECM extract

onto an Immobilon membrane using the slot-blot apparatus. Be sure to run a
vitronectin standard (0.2

µ

g) with this procedure. Incubate the membrane with a
1:1000 dilution of antirabbit and antihuman vitronectin antiserum. Visualize the
immune complex as previously described (11) .

µ

3.4. Preparation of Fibroblast (GM-10) ECM
1. Human fetal dermal fibroblasts (GM-10) are maintained in Eagles Minimal

Essential Media (EMEM, Life Technologies) supplemented with 10% fetal calf
serum (Life Technologies), 110

µ g/mL pyruvate, 30 µ g/mL asparagine, 21 µ g/mL

serine, 100 U/mL penicillin, and 100 µ /mL streptomycin.

2. The cells are plated on positively charged 24 Primaria well plates and grown for

7-10 d until confluence.