Our Zymogram Developing Buffer is used for Running Protein samples for Zymogram PAGE analysis on Polyacrylamide gels. The buffer is supplied as 10X concentration solution and contains 0.5 Tris HCl, 2M NaCl, 50mM CaCl2, 0.2% Brij-35, pH 7.5. Simply dilute it by adding 1/10 volume of 10X Zymogram Developing Buffer to 9/10 volume of DI Water.
Zymography is an electrophoretic technique, which is based on SDS-PAGE and includes an enzyme substrate co-polymerized with polyacrylamide gel. Samples are prepared in Zymogram Sample Buffer without boiling to preserve the structure and activity of the enzyme. Following electrophoresis, the SDS is removed from the gel by washing in Zymogram Renature Buffer that contains a non-ionic detergent. The gels are then equilibrated in Zymogram Developing Buffer, which contains the divalent metal cation calcium, required to activate calcium-dependent proteases such as matrix metalloproteinases (MMPs). The zymogram is subsequently stained (commonly with Amido Black or Coomassie Brilliant Blue), and areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme.