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Gelatine zymography

MB Marcela Bucekova
MS Martin Sojka
IV Ivana Valachova
SM Simona Martinotti
ER Elia Ranzato
ZS Zoltan Szep
VM Viktor Majtan
JK Jaroslav Klaudiny
JM Juraj Majtan
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Conditioned media of HaCaT and HEK cell cultures were subjected to gelatine zymography as previously described44. Briefly, non-reducing LDS sample buffer (Life Technologies, UK) was added to aliquots of culture medium supernatants at a ratio of 1:4. Twenty microlitre aliquots were separated on 8% SDS-PAGE gels containing 0.5 mg/ml gelatine under non-reducing conditions. The gels were washed in 2.5% Triton X-100 for 60 min at room temperature to remove the SDS and were subsequently incubated in a developer buffer [50 mM Tris (pH 7.8), 5 mM CaCl2 and 0.2 M NaCl] for 24 h at 37 °C. Gels were stained with 0.5% Coomassie Brilliant Blue G-250, and the bands of proteolytic activity were quantified by densitometry (Quantity One, Bio-Rad, USA).

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