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 214

5. Wash the plates four times with serum-free DMEM-H. Incubate the plates at

room temperature with 2 mL/plate of 2 M urea (freshly prepared) for about
15-20 min. Check the plates under the microscope: The cells should be visible
against a dark surface. The cells should have a shrunken and collapsed morphology.

6. The media is then aspirated. Use a cell scraper to gently remove the cells from the

plate, and pay special attention to the cells around the edges. It is important to
remove as many cells as possible, but it also important to be gentle enough to
leave the ECM intact.

7. Wash the plate 2-3 times gently with serum-free DMEM-H. Add 0.5 mL 0.5X

Laemmli sample buffer without glycerol to each 10-cm plate. Swirl the plate to
get complete exposure. Remove the ECM with a cell scraper and transfer it to a
1.7-mL Eppendorf microfuge tube. Wash the plate again with 0.5 mL 0.5X
Laemmli buffer to get a second extract, which can be used to test whether or not
the ECM has been successfully extracted with urea.

8. Spin the ECM in a microfuge at 14,000 g for 15 min at -4 C to remove cellular

debris. Measure the protein concentrations and store the samples in liquid nitro-
gen until they are used (preferably less than 3 wk).

°

3.2. 125 I-IGFBP-5 Binding Assay to pSMC ECM
This method is used to determine the binding affinity of ECM proteins for
IGFBP-5 and the capacity of various test substances to alter total IGFBP-5
binding to pSMC/ECM ( see Note 2 ).
1. Dilute the ECM that has been prepared as previously described 1:5 with dis-

tilled water.

2. Prepare a piece of Immobilon P membrane, 11 × 3 cm, and cut a piece of 3M

Whatman filter paper to the same size.

3. Wet the membrane in methanol and wash in 30 m M sodium phosphate containing

0.2% Triton X-100, pH 7.4. Wash the filter paper in the same buffer. Load the
slot blot apparatus with the Immobilon P membrane and filter paper.

4. Load 170 L of diluted ECM into each well. Suction transfer the ECM onto the

membrane. Wash each well with 0.1 M Tris, pH 7.4 and suction transfer again
until the wells are empty.

µ

5. Remove the membrane from the blotting apparatus and allow it to dry on a piece

of filter paper. Wet the blots with methanol and wash in TBS for 5 min at room
temperature. Block the membranes for 5 h at room temperature in TBS contain-
ing 3% BSA. Rinse the membrane in 30 m M sodium phosphate with 0.2% Triton
X-100 twice for 50 min. Cut the membrane into pieces that each contain four
separate ECM extracts.

6. Dilute unlabeled IGFBP-5 with a 30 m M sodium phosphate buffer . The recom-

mended concentrations are 0, 50, 100, 500, and 1000 ng/mL. Add to a final vol-
ume of 2 mL of 30 mM sodium phosphate buffer. If testing mutants, prepare
tubes with identical concentrations. Prepare 125 I-labeled IGFBP-5 (specific
activity 30

g of IGFBP-5, and one
iodobead for 10 min in 0.5 mL of 0.25 M Na 2 PO 4 (pH 7.2). After 10 min at 22

µ Ci/ µ g) by mixing 0.5 mCi of NaI, 2.0 µ

° C,