Activation of MMP
1) Preparation of APMA Solution: In order to prepare APMA solution, initially a stock
solution is prepared which can last for 1 week. The stock solution is prepared as follows:
 Dissolve 3.5 mg APMA (Sigma-Aldrich product no. A9563) in 1 ml of 0.1M
NaOH to prepare 3.5 mg/ml solution at 10mM. These 10mM stocks can be kept
only for 1 week at 4°C
Further dilution of APMA has to be done on the day of the experiment because it is not
stable for long time after dilution.
 The 10mM stock is further diluted 4X with a reaction buffer. The reaction buffer
is typically PBS containing calcium (Sigma-Aldrich product no. D8662)
 Adjust the pH to 7.0-7.5 using 0.1N HCl. The pH is measured using Whatman pH
strips 2629-990. This diluted stock is approximately 2.5mM
 This 2.5mM stock can be used directly to activate MMPs or it can be further
diluted with the reaction buffer to achieve any range of APMA. APMA
concentrations of 0.5-3.0 mM are typical.
2) Activating MMPs:
 Take 1 part of the above stock solution and add it to 9 parts of MMP(Chemicon
product no. cc1031)
 Incubate the mixture at 37°C for 30 minutes. The final solution can be used
directly without dialyzing away the APMA.
 Activation times will vary depending samples and type of MMP. MMP-1 & 9
often require longer times (3-5 hours typical) Optimal activation times must be
determined empirically
NOTE: APMA is not stable thus diluted stock should be made fresh. APMA solutions
higher than 20mM tend to precipitate out when added directly to reactions thus prediluting as detailed above is recommended.
Precautions: Since APMA is a mercuric compound it is highly toxic and dangerous for
the environment. Therefore one has to be very careful while using it. APMA should
always be used inside a chemical fume hood. A Half-Facepiece Reusable Respirator
(Fisher Cat no. 17-986-9B) with cartridge filter inserts (Fisher Cat no.18-306) and gloves
should be used while handling APMA to avoid inhalation and skin contact. In case of a
spill mercury spill kit (Fisher Cat no. 18-999-948) should be used.