Zymmography gels - (Oct/12/2001 )
I'm having poor results with my zymmography gels. I run the gel for 45min at 100v in -4C. Then I incubate in 2.5% Triton-X and Tris solution for a half an hour and then 1% Triton X over night. The Tris solution consists of tris base, CaCl with HCl to adjust the pH to 7.4 and 1mL of a ZnCl solution (13.6mg of ZnCl in 10mL of water). I stain for three hours and then destain for as long as necessary. I can see where the collagenase has eaten away at the gel but it isn't as clear as it should be. Any suggestions.
1. Run the electrophoresis as normal SDS-PAGE at 4C (for minigel: 10mA stacking and 20 mA running gels, about 2 hrs).2. incubate the gel in 2.5% Triton for 30 min with gentle shaking. Repeat it. Wash with distilled water3. incubate with your reaction buffer at room/37C temperature for ?? hr (hr depends on the conc/specificity of your enzyme, which should be decided by doing several zymograms.4. Stain with CBB 250 and destain with 10% AcOH (acetic acid) only.
Hope this will work. If it does not work, then try to change the reaction buffer. Wish you a good success.