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A study of protein synthesis in subpopulations of cultured human epidermal keratinocytes using two‐dimensional gel electrophoresis
Author(s) -
Easty David J.,
Patel Ketan,
Dover Robin,
Dunn Michael J.,
Evans David J.
Publication year - 1987
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150080807
Subject(s) - percoll , epidermis (zoology) , differential centrifugation , in vitro , keratinocyte , microbiology and biotechnology , biology , gel electrophoresis , in vivo , centrifugation , chemistry , biochemistry , anatomy
A procedure for the isolation of protein markers of epidermal differentiation in vitro is described. Human epidermal keratinocytes were cultured and radiolabelled in vitro . Fractionation was performed according to buoyant density (which reflects the degree of terminal differentiation) using Percoll density gradient centrifugation. Subpopulations of keratinocytes were characterised using light and electron microscopy, and proteins fractionated using high resolution two‐dimensional gel electrophoresis. Radio‐labelled proteins were detected using autoradiography and fluorography. Integral membrane proteins were characterised using Triton X‐114 phase shift extraction. Data from this in vitro study were compared to silver stained gels of samples from intact epidermis ( in vivo ). We report quantitative differences between 14 specific protein moieties expressed in subpopulations of keratinocytes and identify some of these proteins. The differential expression of these protein markers and their possible use in the interpretation of the keratinocyte maturation pathway in cultured cells from patients with skin diseases are discussed.