6533b853fe1ef96bd12ac2f7

RESEARCH PRODUCT

Staining SDS-PAGE gels of skeletal matrices after western blot: a way to improve their sharpness.

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7 pages; International audience; Denaturing 1D electrophoresis on acrylamide gels - also referred as SDS-PAGE - is a classical technique for fractionating and visualizing the macromolecular constituents of matrices associated to calcified tissues. This technique has been widely used in association with the subsequent silver nitrate staining. But because matrices associated to calcified tissues are very often glycosylated and constituted of numerous polydisperse macromolecules, the obtained pattern is frequently 'smeary' and discrete bands, when present on the gel, are often blurred, thickened or totally masked by the polydisperse macromolecules. In this paper, we present a simple protocol that can circumvent this drawback and 'clean' the gels. In short, after the classical migration step of the matrix macromolecules, the gel is transferred (electro-blotting) on a PVDF membrane, similarly to a Western blot, but for a shorter time (partial transfer, i.e., one hour or less). The gel is subsequently stained with silver nitrate. The likely effect of the transfer is to partly remove polydisperse macromolecules and to 'sharpen' the discrete bands. We think that this extra-step may improve in several cases the gel pictures of skeletal matrix components. We illustrate this phenomenon with two examples taken from brachiopod and mollusc shell matrices.