Journal of Protein Chemistry

, Volume 20, Issue 4, pp 273–278

Purification and Partial Amino Acid Sequence of Proteins from Human Epidermal Keratinocyte Conditioned Medium

  • Aftab Ahmed
  • Paul Kandola
  • Gina Ziada
  • Nancy Parenteau
Article

Abstract

Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO3) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.

Skin keratinocyte tissue culture conditioned medium chromatography amino acid sequencing 

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Copyright information

© Plenum Publishing Corporation 2001

Authors and Affiliations

  • Aftab Ahmed
    • 1
  • Paul Kandola
    • 1
  • Gina Ziada
    • 1
  • Nancy Parenteau
    • 1
  1. 1.DevelopmentOrganogenesis Inc. Research &Canton

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