Simplified purification procedure of laminin-332 and laminin-511 from human cell lines

Biochem Biophys Res Commun. 2008 Oct 24;375(3):410-3. doi: 10.1016/j.bbrc.2008.08.029. Epub 2008 Aug 17.

Abstract

Laminins are glycoproteins expressed in the basement membrane of multiple epithelial tissues. Previously described purification procedures for the human laminin variants laminin-5 (LN-332) and laminin-10 (LN-511) use tissue as starting material and have multiple steps. We demonstrate a two-step laminin immunoaffinity purification method to produce consistent quantities of intact and biologically active LN-332 and LN-511 from human keratinocyte (HaCaT) and human lung carcinoma (A549) cell lines, respectively. The purification of LN-332 and LN-551 was demonstrated by PAGE analysis, silver staining and Western blot analysis. The purification procedure includes instruction on removing a cell adhesion contaminant known as galectin-3 binding protein from purified LN-511. The biological activity of purified laminin was tested in a standard cell adhesion assay and compared to commercially available LN-111. This rapid and reproducible purification method will contribute to understanding the role of LN-332 and LN-511 in cell behavior, signaling, and gene expression.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibody Affinity
  • Cell Adhesion Molecules / immunology
  • Cell Adhesion Molecules / isolation & purification*
  • Cell Line
  • Chromatography, Affinity / methods*
  • Culture Media, Conditioned / chemistry
  • Humans
  • Keratinocytes / chemistry
  • Laminin / immunology
  • Laminin / isolation & purification*

Substances

  • Cell Adhesion Molecules
  • Culture Media, Conditioned
  • Laminin
  • kalinin
  • laminin 10