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 212

In summary, IGFBP-5 association with ECM is an important means of
potentiating the cellular response to IGF-I. Sequestering large amounts of IGF-I
within the ECM may be an important component of the response to injury
when an advanced proliferative activity under IGF-I stimulation is required
(7) . Likewise, during normal, balanced growth, provision of large quantities of
IGFBP-5 in the ECM could provide an important source of growth factor dur-
ing times of decreased synthesis, when balanced growth is still required. Fur-
thermore, because epithelial cells do not have a large amount of ECM,
connective-tissue-cell matrix may provide an important source of IGF for
regenerative-epithelial tissue that is rapidly dividing, such as gastrointestinal
epithelium. The techniques that have been worked out to isolate ECM (8) and
then quantify IGF binding to ECM that are detailed below have been instru-
mental in proving these points.
Under specialized circumstances, IGFBP-2 has also been shown to associ-
ate with ECM. Preparation of ECM and layering of IGFBP-2 shows that it is
not preferentially bound. However, if high concentrations of IGF-I are added
that result in a confirmational change in IGFBP-2, this altered conformation
can bind to ECM. This may be important in focally concentrating large amounts
of IGF in ECM of tissues that synthesize IGFBP-2. Because IGFBP-2 is an
important product of epithelial cells, and because epithelial-cell basement
membranes often contain IGFBP-2, this may be a particularly important means
for focally concentrating IGFs near epithelial tissue. This has been shown to be
very important in the olfactory tissue within the brain, wherein large amounts
of IGF-I and IGFBP-2 are synthesized and large amounts can be seen focally
concentrated in the ECM of glial cells.

2. Materials
2.1. Preparation of Porcine Smooth Muscle ECM
1. Dulbecco's modified Eagle's medium with high glucose (DMEM-H) (Life Tech-

nologies, Grand Island, NY).

2. Fetal bovine serum (FBS) (Life Technologies).

3. Cell scrapers.

4. Laemmli sample buffer without glycerol.

2.2. 125 I-IGF BP-5 Binding Assay to pSMC ECM
1. Immobilon membranes (Millipore, Bedford, MA).

2. 30 m M sodium phosphate containing 0.2% Triton X-100, pH 7.4.

3. Slot blotter apparatus model 2643 from Bethesda Research Laboratories

(Gaithersburg, MD).

4. 0.1 M Tris, pH 7.4.

5. Methanol.