QUANTIFICATION OF AG-NOR PROTEINS USING AG-NOR STAINING ON WESTERN BLOTS

Ribosomal genes are associated with a set of silver-stained nucleolar proteins, the Ag-NOR proteins, whose amount is directly related to the duration of the cell cycle. Quantification of Ag-NOR proteins by imag e analysis is presently used to evaluate the rate of proliferation of cancer cells and nucleolar activity. Our objective was to establish a procedure to quantify independently each major Ag-NOR protein in cell extracts. Computerized densitometry established that the specific silv er staining of Ag-NOR proteins (Ag-NOR staining) performed on Western blots makes it possible to quantify Ag-NOR proteins. Using purified Ag -NOR proteins, nucleolin, and protein B23, we observed that the intens ity of Ag-NOR staining is proportional to the amount of protein. A lin ear relationship exists between the intensity of Ag-NOR staining and t he amount of nudeolin, in the range of 0.2-1.6 mu g. Using total nucle ar extracts prepared from mammalian cells, the proportionality was mai ntained for total Ag-NOR-stained proteins or for a particular protein. We also determined the levels of nuclear proteins suitable for quanti tative analysis. Individual Ag-NOR proteins can be quantified by compu terized densitometry in nuclear extracts after Ag-NOR staining on West ern blots. This procedure can be applied to establish the contribution of each Ag-NOR protein in general staining, estimate the variability of each Ag-NOR protein in normal and pathological conditions, and quan tify each Ag-NOR protein contained per cell.