Selective surface radioiodination of keratinocytes in primary culture labels a 59 kD keratin and other surface proteins.

Archives of Dermatological Research
E M DuganL A Diaz

Abstract

Murine keratinocytes were vectorially labeled in order to identify and characterize surface proteins. Freshly trypsinized keratinocytes were obtained from neonatal BALB/c mice and primary cultures were established. Cells were radiolabeled with iodogen and 125I immediately after harvesting and after 4, 24, 48, and 72 h in culture. Cells were solubilized with 1% sodium dodecyl sulfate (SDS) and reducing agents (total extract), or sequentially extracted with: (a) 1% Triton X-100 (membrane/cytosol fraction); (b) 2 M NaCl (salt fraction); and (c) 1% sodium dodecyl sulfate (SDS; cytoskeleton fraction). Extracts were analyzed by 1- or 2-D polyacrylamide gel electrophoresis (PAGE), followed by autoradiography (AR). The 59 kD acidic murine keratin was identified by immunoblot analysis with monoclonal antibody AE1 and a human polyclonal anti-59 kD keratin autoantibody designated Cascas-42. Total extracts contained up to ten labeled proteins ranging from 10 to 180 kD. Eight of these proteins were present in the membrane/cytosol fraction (10, 12, 18, 30, 38, 41, 66, and 130 kD). Intense labeling of a 59 kD cytoskeletal protein was consistently seen; this protein was identified as the 59 kD acidic murine keratin (Moll's catalogue no. 10) by...Continue Reading

References

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Citations

Sep 25, 2007·Applied Radiation and Isotopes : Including Data, Instrumentation and Methods for Use in Agriculture, Industry and Medicine·F Z Biber MuftulerY Yürekli

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