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| This protocol measures Matrix Metalloproteinase (MMP-1) activity of conditioned media. MMPs are proteolytic enzymes capable of degrading connective tissue components such as collagen. |
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A. Coating of Wells with Radiolabeled Collagen
1. Use 14C-acetylated collagen (see Protocol on Preparation of Radiolabeled 14C-Acetylated Collagen). Add 20 μl of the 14C-acetylated collagen to the required number of wells in a 96-well microtiter plate (see Hint #1)
2. Incubate the plate at a 45( angle for 6 hr at 37°C.
B. Activation of Procollagenase (see Hint #2)
1. Mix 100 μl of serum-free conditioned media and 100 μl of 1X Trypsin. Dilute the trypsin from the Trypsin Stock in Assay Buffer.
2. Incubate for 30 min at room temperature (see Hint #3).
3. Stop the trypsinization by adding 50 μl of 1X Soybean Trypsin Inhibitor. Dilute the Soybean Trypsin Inhibitor Stock in Assay Buffer.
C. Collagenase Assay
1. Add 200 μl of the activated media to wells of the 96-well plate.
2. Set up some wells as negative controls lacking the activated collagenase. Add 200 μl of Bacterial Collagenase at a concentration of 10 ng/ml to some wells as a positive control. Dilute Bacterial Collagenase Stock to 1X in Assay Buffer.
3. Incubate the plate overnight at 37°C.
4. Remove the supernatant from each well and add it to scintillation vials, along with the scintillation fluid.
5. Count the samples in a scintillation counter.
6. Calculate the MMP-1 activity for each sample
1 unit MMP-1 activity = degradation of 1 μg collagen/min at 37°C
specific activity of collagen = cpm/μg input collagen
Units MMP-1 activity = [(sample cpm - background cpm)/specific activity of collagen)] x (dilution factor)/(min)
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| Trypsin Stock (100X) |
| Store aliquots at -20°C
Prepare in Assay Buffer
1 mg/ml Trypsin
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| Assay Buffer |
| 100 mM Tris-Cl, pH 7.5
2 mM CaCl2
Autoclave
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| Bacterial Collagenase Stock (100X) |
| Store aliquots at -20°C
Prepare in Assay Buffer
1 μg/ml Collagenase
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| Soybean Trypsin Inhibitor Stock (20X) |
| 6 mg/ml Soybean Trypsin Inhibitor
Prepare in Assay Buffer
Store aliquots at -20°C.
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Bacterial Collagenase
Soybean Trypsin Inhibitor
Media, Conditioned, Serum-free
Trypsin
Tris
Calcium Chloride
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The incubation time and amount of conditioned media to add can be adjusted to accommodate your specific assay parameters.
3. The Trypsin cleaves the Procollagenase in the conditioned media to Collagenase, thus activating the enzyme.
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