Collagenase, Type I, Clostridium histolyticum - FAQs

For cell digestion, we recommend preparing collagenase using a salt solution such as Hank's Balanced Salt or Earle's Balanced Salt solution at concentrations in the range of 0.1-5 mg/mL. BSA (0.5%) or serum (5-10%) can be added to stabilize cells during digestion.

For assaying enzymatic activity using Collagenase, Type I, Clostridium histolyticum (Cat. No. J62406.xx), we recommend the following procedure:

1. Prepare enzyme stock solution. Create a stock solution by dissolving enzyme at a concentration of 1 mg/mL in 0.05 M TES with 0.36 mM calcium chloride, pH 7.5.
2. Prepare enzyme working dilutions for assay. We recommend making either a 1/10 dilution or a 1/20 dilution of the stock solution above.
3. Weigh 25 mg of Collagenase, Type I, Clostridium histolyticum into each of four test tubes. Include at least two tubes to serve as blanks. The blanks will not contain enzyme. Add 5.0 mL of 0.05 M TES buffer to the tubes (including the blanks) and incubate at 37°C for 15 minutes.
4. Start the reaction by adding 0.1 mL of enzyme dilution to appropriate tubes. For blanks, substitute 0.1 mL of enzyme dilution with 0.1 mL of 0.05 M TES buffer.
5. After 5 hours, stop the collagenase reaction by transferring 0.2 mL of solution from the experimental or blank test tubes to tubes containing 1.0 mL of ninhydrin-citric acid mixture. Make sure not to transfer over the collagen.
6. Heat the test tubes for 20 minutes in a boiling water bath and then let them cool.
7. After cooling, dilute with 5 mL of 50% n-propanol and let stand for 15 minutes.
8. Read absorbance at 600 nm and use an L-leucine standard curve to determine micromoles amino acid equivalent to leucine liberated.

We recommend storing lyophilized powder at 2-8°C. Solutions should be stored at -20°C in aliquots to limit the number of freeze/thaw cycles. Freeze-thaw cycles will damage the enzyme solution.

Activators: Ca2+ and Zn2+ are collagenase activators.
Inhibitors: EDTA, EGTA, DTT, β-mercaptoethanol, o-phenanthroline and glutathione (reduced) are collagenase inhibitors.