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 216

3. Aspirate the media and rinse the plates twice with PBS.

4. Incubate the cells with 0.3 mL lysis buffer for 2-5 min until the cells lyse and

nuclei are no longer apparent by microscopy. Usually this does not take more
than 5 min. Aspirate the lysis buffer, gently rinse the cells twice with PBS,
and aspirate.

5. Incubate the cells in each well with 0.3 mL of 25 m M amonium acetate, pH 9.0,

for 3-5 min to remove the remaining nuclei and cytoskeletal elements. Observe
the plates microscopically and do not extract for too long a time period.

6. Add 3 drops 1 M HEPES, pH 7.3, to neutralize pH and then aspirate. Gently rinse

the cells twice with PBS, once with 30 mM sodium phosphate buffer with 1%
BSA, pH 7.4.

7. Check the plate under the microscope. Intact ECM should be visible, densely

striated with some degree of variation.

To avoid matrix lifting, use cold reagents and keep ECM on ice at all times.

3.5. Binding of 125 I-IGFBP-5 or Unlabeled IGFBP-5
to Fibroblast ECM
The purpose of this method is to determine the binding of IGFBP-5 to fibro-
blast ECM, to quantify the ability of the substances to compete with IGFBP-5
for binding, and to determine the effect of test substances added to cultures to
alter the binding of IGFBP-5 to ECM.
1.

Prepare ECM according to the previous protocol. Add unlabeled IGFBP-5 pep-
tide in increasing amounts (0.04-2.0 µ g/mL in 30 m M sodium phosphate buffer,

0.1% BSA, pH 7.4, total volume 500 L) to duplicate culture wells. Incubate on

ice for 30 min to allow IGFBP-5 to bind to ECM. Add 250,000 cpm of IGFBP-5
prepared as described previously to each well. After 14 h at 4

µ

C with gentle
agitation, aspirate the media and rinse each well with 30 mM sodium phosphate
buffer twice.

°

2. Extract the ECM off the plate with 0.5 mL of 0.3 N NaOH twice and determine

the amount of 125 I-IGFBP-5 directly by gamma counting. In the experiments
measure the capacity of unlabeled IGFBP-5 to bind to the ECM, prepare the
fibroblast ECM extracts as described above, add the substances to be tested to
duplicate wells containing 0.5 L of the same buffer described previously. Add

40 ng of unlabeled IGFBP-5 to each well and incubate overnight. Wash the plates
as described previously. Extract the ECM with 50

µ

L of 1X Laemmli sample
buffer. Remove insoluble material by centrifuging at 15,000 g for 10 min. Heat
the supernatants to 95

µ

° C for 10 min.

3. Electrophorese the supernatants through 12.5% SDS polyacrylamide gel, trans-

fer to Immobilon PDQ (Millipore) membranes, and then analyze the protein
extracts by Western ligand blotting, using 125 I-IGF-I. Determine band intensity
by Phosphor Image analysis (Molecular Dynamics, Sunnyvale, CA). To confirm
that the band that binds 125 I-IGF-I is IGFBP-5, the extracts can be analyzed by
immunoblotting using a 1:1000 dilution of anti-IGFBP-5 antiserum.