Quantitation of cross-linked protein: an alternative to counting cornified envelopes as an index of keratinocyte...

Covalently cross-linked protein is the key feature of the cornified envelope, an important marker of squamous differentiation. Enumeration of cornified envelopes is widely used to assess keratinocyte differentiation, but is tedious and subjective. We report here a rapid, objective, sensitive, and quantitative assay that measures total cross-linked protein in keratinocytes. The method is based on the resistance of cross-linked protein to solubilization in boiling SDS-beta ME, separation of cross-linked from soluble protein by collection of cross-linked protein on sheets of regenerated cellulose, binding of Coomassie blue to protein, and quantitation by scanning laser densitometry. There is excellent correlation between the cross-linked protein measured by this assay and the number of cornified envelopes.