Dot blot: Serum-free conditioned medium (DMEM) from SH-SY5Y cells, cultured for 72h, was spotted onto nitrocellulose membrane using a dot blot apparatus. CSPG mix (Millipore) diluted with DMEM were used as a positive control. Half the samples from each group were treated with ChABC for 3h, prior to incubation of the membrane with antibody 2B6 (mouse monoclonal to epitopes exposed by ChABC digestion) 1:500, Seikagaku. This detects a stub epitope exposed by ChABC digestion of CSPGs. The membrane was then washed and incubated with HRP anti-mouse IgG 1:10,000. The signal was detected using chemiluminescence (Luminata) and visualised using hyperfilm (Amersham).
CSPG assay: A Blyscan sulphated glycosaminoglycan assay (Biocolor) [24 (link)], was used to detect the presence of glycosaminoglycan chains in the conditioned medium of differentiated SH-SY5Y cells. This dye binding assay is a quantitative measure of intact CSPG GAG chains. A CSPG mix (Millipore) was used as a positive control. Absorbance was measured at 656nm and sulphated-glycosaminoglycan concentrations obtained from a standard curve.
Day P., Alves N., Daniell E., Dasgupta D., Ogborne R., Steeper A., Raza M., Ellis C., Fawcett J., Keynes R, & Muir E. (2020). Targeting chondroitinase ABC to axons enhances the ability of chondroitinase to promote neurite outgrowth and sprouting. PLoS ONE, 15(1), e0221851.
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