Biology Reference
In-Depth Information
4. Notes
1.
Experience gained with these technologies has shown that several steps in the
methodology require close observation in order to obtain optimal results. In pre-
paring fibroblast ECM, the monolayers need to be observed during the removal
of cells and cytoskeletal elements. Failure to observe the cultures microscopi-
cally will result in either inadequate removal of cell bodies and nuclei or exten-
sive removal such that most of the ECM is lost. This technique is an
approximation at best, and therefore one is always attempting to strike a balance
between adequate removal of the non-ECM components and retention of suffi-
cient ECM to complete the studies. The aim is to be able to obtain a preparation
of ECM that retains a protein composition that is similar to the native ECM as
well as protein folding and conformational states such that binding activities are
not completely destroyed. The success to which this can be undertaken is depen-
dent upon the amount of ECM produced per cell. For example, in tissues such as
fibroblasts in culture, there is a large amount of ECM produced per cell, so a
large amount of matrix can be obtained with successful removal of cell bodies
and cytoskeletal elements. However, for smooth muscle cells, there is much
less matrix produced per cell; therefore, no method will entirely remove all the
cells without removing some of the ECM, and the cultures need to be observed
during the extraction procedure. In summary, one must always strike a balance
between removal of cellular material and retention of ECM. This can only be
done by close, microscopic observation during the extraction procedures.
2.
In some cases, such as analysis of binding properties of smooth muscle cell ECM
proteins, it may be necessary to perform the binding assays using a matrix other
than a plastic culture dish. The reason for this is that all tissue culture plastic is
prepared with highly charged polyanionic substances in order to facilitate cell
attachment. These charged substances will bind proteins that are added exog-
enously quite avidly. Therefore, if a large quantity of ECM is present, it will
nearly cover and neutralize the polyanionic charges. However, if part of the ECM
is removed during cell removal, such as in preparation of smooth muscle cell
ECM, then the nonspecific binding to the tissue culture plastic may be so great
that one cannot accurately quantify binding to ECM. In order to obviate this prob-
lem, we developed a method of filter binding assays. While this method accu-
rately quantifies binding to specific ECM proteins, it does not allow maintenance
of these proteins in their natural, three dimensional conformation in which they
are maintained in the ECM, since they must be extracted completely and then
readhere to the membranes. However, using this technology, one can block non-
specific binding to the filter by pretreating the filters with high concentrations of
BSA. Therefore, one can definitively demonstrate valid binding to ECM proteins
by showing that only the slot that is blotted on the filter with ECM proteins will
bind to a radiolabeled antigen (in our case radioiodinated IGFBP-5). Therefore,
this methodology gives an approximation of the amount of ECM protein that is
present and the binding capacity of those proteins that can be extracted for a
particular radiolabeled substrate. Furthermore, it is quite is easy to correct for
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