Improved methods for separation of human cytokeratins

B. Meiklejohn, Raymond B Nagle, K. M. McDaniel, G. S. Wilson

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

The cytokeratins from human bladder and esophageal epithelia were separated using chromatographic techniques. The cytokeratins were first extracted from fresh autopsy tissue using high and low salt buffers. Urea, 8.0-9.5 M, was used to solubilize the resulting cytokeratin pellet. Imidazole was found to increase the solubility of the pellet but reducing agents such as 2-mercaptoethanol were not beneficial. DEAE ion exchange chromatography produced three fractions which were analyzed by using one and two-dimensional electrophoresis. The third fraction was shown to contain the acidic cytokeratins and was further fractionated on a moderately polar reverse phase HPLC column using an acetonitrile elution gradient. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase, and trifluoroacetic acid was added to ion pair with the protein. HPLC fractions of the acidic proteins from human esophagus revealed seven reproducible peaks. All seven peaks were shown by Western blotting to contain an epitope found on cytokeratin 13. The results suggest that the isolation and separation procedures have produced a series of peptide products which all retain a similar epitope but which vary significantly in their hydrophobic characteristics.

Original languageEnglish (US)
Pages (from-to)637-645
Number of pages9
JournalLife Sciences
Volume47
Issue number7
DOIs
StatePublished - 1990

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Keratins
Epitopes
Keratin-13
High Pressure Liquid Chromatography
Trifluoroacetic Acid
Mercaptoethanol
Reducing Agents
Ion Exchange Chromatography
Chromatography
Electrophoresis
Solubility
Esophagus
Urea
Autopsy
Ion exchange
Buffers
Urinary Bladder
Proteins
Epithelium
Salts

ASJC Scopus subject areas

  • Pharmacology

Cite this

Improved methods for separation of human cytokeratins. / Meiklejohn, B.; Nagle, Raymond B; McDaniel, K. M.; Wilson, G. S.

In: Life Sciences, Vol. 47, No. 7, 1990, p. 637-645.

Research output: Contribution to journalArticle

Meiklejohn, B, Nagle, RB, McDaniel, KM & Wilson, GS 1990, 'Improved methods for separation of human cytokeratins', Life Sciences, vol. 47, no. 7, pp. 637-645. https://doi.org/10.1016/0024-3205(90)90576-D
Meiklejohn, B. ; Nagle, Raymond B ; McDaniel, K. M. ; Wilson, G. S. / Improved methods for separation of human cytokeratins. In: Life Sciences. 1990 ; Vol. 47, No. 7. pp. 637-645.
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