A simple procedure for tenascin purification.
Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.
Saginati
M
M
Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Siri
A
A
Balza
E
E
Ponassi
M
M
Zardi
L
L
D015816:Cell Adhesion Molecules, Neuronal / Q000302:isolation & purification*; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000032:analysis; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D003470:Culture Media; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification*; D006801:Humans; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D008545:Melanoma; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification; D019063:Tenascin
D015816:Cell Adhesion Molecules, Neuronal; D003470:Culture Media; D016326:Extracellular Matrix Proteins; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D019063:Tenascin; D002809:Chondroitin Sulfates; D017886:Durapatite