Pronase streptomyces griseus

Fresh human tissue was washed in PBS, frozen, and lyophilized. The dried tissue was crushed into a fine powder, weighed, resuspended in PBS containing 1 mg/mL Pronase (Streptomyces griseus, Sigma Aldrich) and 0.1% Triton X-100, and incubated at 37°C overnight with shaking. The samples were centrifuged at 20,000 x g for 20 min and the supernatant was mixed 1:10 with equilibration buffer (50 mM sodium acetate, 0.2 M NaCl, 0.1% Triton X-100, pH 6) and loaded onto a DEAE Sephacel column (GE healthcare) equilibrated with buffer. The column was washed with 50 mM sodium acetate buffer containing 0.2 M NaCl, pH 6.0, and bound GAGs were eluted with 50 mM sodium acetate buffer containing 2.0 M NaCl, pH 6.0. The eluate was mixed with ethanol saturated with sodium acetate (1:3, vol/vol) and kept at −20°C overnight, followed by centrifugation at 20,000 x g at 4°C for 20 min. The pellets were dried in a centrifugal evaporator and reconstituted in DNase buffer (50 mM Tris, 50 mM NaCl, 2.5 mM MgCl₂, 0.5 mM CaCl₂, pH 8.0) with 20 kU/mL bovine pancreatic deoxyribonuclease I (Sigma Aldrich) and incubated with shaking for 2 h at 37°C. The samples were adjusted to 50 mM Tris and 50 mM NaCl, pH 8.0, and incubated for 4 h at 37°C with 20 mU/mL chondroitinase ABC (Proteus vulgaris, Sigma Aldrich). The HS was purified over a DEAE column and precipitated with 90% ethanol (Esko, 1993 ).