Differential staining of collagens and non-collagens with Coomassie Brilliant Blue G and R.
The following proteins were subjected to electrophoresis in SDS gels and stained with both Coomassie Brilliant Blue R and Coomassie Brilliant Blue G: the pepsin-treated collagen types I, II, III and V, and non-pepsin-treated type IV collagen, and the non-collagens, laminin, fibronectin, myosin, beta-galactosidase, fibrin, phosphorylase b and serum albumin. The Coomassie Brilliant Blue G stain was formulated as in the dye-binding protein assay reagent of Bradford (Anal. Biochem. 72: 248-254, 1976). Coomassie Brilliant Blue R prominently stained all polypeptides, but the collagen chains, including the type IV chains, stained metachromatically (red or pink) while the non-collagens stained orthochromatically (blue-violet). In the Bradford reagent, however, only the non-collagens and the intact type IV chains were prominently stained; the pepsin-treated collagen chains were virtually undetectable provided that detergent had been exhaustively removed prior to immersion in the stain. Metachromatic staining with Coomassie Brilliant Blue R is attributed to the presence of closely-spaced proline and hydroxyproline residues in sequences from triple-helical domains. The staining of type IV chains with the Bradford reagent is attributed to the presence of binding sites in the sequences from the non-triple-helical domains only, since such binding sites are absent from chains derived from the pepsin-treated collagens.