Simplified purification procedure of laminin-332 and laminin-511 from human cell lines.
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.
Sroka
Isis C
IC
Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724, USA.
Chen
Man Ling
ML
Cress
Anne E
AE
D000915:Antibody Affinity; D015815:Cell Adhesion Molecules / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D017077:Culture Media, Conditioned / Q000737:chemistry; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry; D007797:Laminin / Q000276:immunology / Q000302:isolation & purification*; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D017077:Culture Media, Conditioned; D007797:Laminin; D000097794:Kalinin; C119013:laminin 10