I have repeatedly attempted to activate the proenzyme form of MMP-1 and MMP-8 by incubating them in 1 mM APMA, but to no success. According to the standard protocol, MMP-1 and MMP-8 are activated by incubating them at 37°C in 1mM APMA for 3 hr and 1 hr, respectively. The APMA (Sigma-Aldrich, A9563) stock was made at a concentration of 10 mM using 0.6 M NaOH solution and added to the MMP stock solutions, diluting the APMA to the desired concentration. However, upon running SDS-PAGE we are unable to see any cleavage of the proenzymes. Am I missing something? Any suggestions? Thank you in advance.
Article An improved collagen zymography approach for evaluating the ...
Article Activation of recombinant human neutrophil procollagenase in...
Are you concerned that the final pH might be extremely high due to dissolving the APMA in 0.6 M NaOH. The enzymes might be denatured by the high pH. I recommend trying again, but instead dissolving the APMA in buffer or DMSO.
I agree with that approach. I use 1 mM APMA to activate 1 micromolar of proMMPs at 37 degrees, 1 hour from a 100 mM APMA stock in 100% DMSO.
Thanks everyone for your advice and suggestions. I'll definitely give the DMSO a shot.
- Badges
- Science method