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 218

nonspecific binding using this technology, and therefore the residual binding that
can be measured is generally a valid assay. Attempts should be made during the
binding assay to use appropriate concentrations of divalent cations, salt, and so
on, to approximate physiologic conditions that would be present in the normal
ECM in situ . Again, some assurance that the extracted matrix represents the true
matrix as deposited by the cells can be obtained by comparing ratios of different
matrix proteins within the extract to determine that they approximate the ratios of
those proteins that are present in situ as the matrix is synthesized.

3. When conducting binding studies in situ using tissue culture plates, the remain-

ing ECM after tissue extraction is often densely adherent to the plate. Therefore,
in addition to adding harsh detergents such as SDS, one often needs to scrape the
plate with a mechanical scraper in order to remove the remaining ECM proteins.
To ensure that removal is complete, a reasonable technique is to re-extract the
plate with a high concentration of SDS and then conduct immunoblotting for a
common matrix protein such as vitronectin. This will ensure that the method is
adequate for complete extraction of the proteins of interest.

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