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C0130 Sigma

Collagenase from Clostridium histolyticum

for general use, Type I, 0.25-1.0 FALGPA units/mg solid, ≥125 CDU/mg solid

Synonym: Clostridiopeptidase A

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Properties

Related Categories 3.4.x.x Peptidases, 3.x.x.x Hydrolases, Application Index, Biochemicals and Reagents, Cell Dissociation,
form   essentially salt-free, lyophilized powder
mol wt   mol wt 68,000 to 125,000
optimum pH   6.3-8.8
solubility   TESCA buffer (50 mM TES, 0.36 mM Calcium chloride, pH 7.4): soluble1-2 mg/mL at 37 °C
storage temp.   −20°C

Description

Frequently Asked Questions

Frequently Asked Questions are available for this Product.

Biochem/physiol Actions

Collagenase is activated by four gram atom calcium per mole enzyme. It is inhibited by ethylene glycol-bis(beta-aminoethyl ether) - N, N, N′,N′-tetraacetic acid, beta-mercaptoethanol, glutathione, thioglycolic acid and 8-hydroxyquinoline.

The collagenase product is a mixture of enzymes secreted by C. histolyticum, with different products differentiated by the relative ratios of the 10-18 components found in the secreted enzymes. The main components are two collagenases, clostripain, and a neutral protease. The synergistic action of these enzymes degrade collagen and other intracellular materialThe action of both collagenase enzymes and the neutral protease is necessary for effective release of cells from tissue. Various types of collagen are the natural substrates for collagenase.

Caution

As supplied, this product is stable for one year at -20°C. There is no loss in FALGPA or protease activity in 30 days at 37°C, 50°C and -20°C. Solutions of crude collagenase are stable if frozen quickly in aliquots (at 10 mg/mL) and kept frozen at -20°C. Further freeze-thaw cycles will damage the solution. The product retains 100% activity over 7 hours when held on ice.

Packaging

1, 5 g in poly bottle

100, 500 mg in poly bottle

Preparation Note

This product is an equivalent to first 40% ammonium sulfate fraction of Mandl, I., et al., J. Clin. Invest., 32, 1323 (1953). Solutions are typically prepared at 1-2 mg/mL in TESCA buffer (containing 50 mM TES, 0.36 mM Calcium chloride, pH 7.4 at 37°C.

Unit Definition

One collagen digestion unit (CDU) liberates peptides from collagen from bovine achilles tendon equivalent in ninhydrin color to 1.0 μmole of leucine in 5 hours at pH 7.4 at 37 °C in the presence of calcium ions. One FALGPA hydrolysis unit hydrolyzes 1.0 μmole of furylacryloyl-Leu-Gly-Pro-Ala per min at 25°C. One Neutral Protease unit hydrolyzes casein to produce color equivalent to 1.0 μmole of tyrosine per 5 hr at pH 7.5 at 37°C. One Clostripain Unit hydrolyzes 1.0 μmole of BAEE per min at pH 7.6 at 25°C in the presence of DTT.

Application

Collagenase from Clostridium histolyticum has extensive use in biological studies, where it is used to prepare isolated cell suspensions. This product is suitable for the disaggregation of human tumor, mouse kidney, human adult and fetal brain, lung and many other epithelia tissues. It has also been shown to be effective in liver and kidney perfusion studies, digestion of pancreas, isolation of nonparenchymal rat liver cells and hepatocyte preparation. Collagenase has also been used in the preparation of arterial tissue for the study of Advanced Glycosylation End Products. This enzyme has been tested for the release of heptatocytes at a concentration of approximately 1 mg/mL. Concentrations for digestion range from 0.1 to 5 mg/mL.

General description

Collagenase is a protease which cleaves the triple-helical protein called collagen. There are three types of tissue collagenases, and these belong to the matrix metalloproteinases (MMP) family. Collagenase obtained from Clostridium histolyticum has a very strong activity, as it digests collagen from both ends, at temperatures as low as 4-10°C. Crude collagenase mixtures contain two major enzyme types namely, collagenase and clostripain.

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Biomedical Applications
Safety & Documentation

Safety Information

Symbol 
Signal word 
Danger
Hazard statements 
Precautionary statements 
Personal Protective Equipment 
RIDADR 
NONH for all modes of transport
WGK Germany 
1

Frequently Asked Questions

How should I prepare a stock solution of Product C0130, Collagenase?
Stock solutions of crude Collagenase may be prepared at 10 mg/ml of Collagenase in TESCA buffer (50 mM TES, 0.36 mM calcium chloride, pH 7.4 at 37°C) and are stable if frozen quickly in aliquots and kept frozen at -20°C. Freeze-thaw cycles will damage the enzyme. To view this information on the C0130 data sheet click on the following link:   http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/c0130pis.Par.0001.File.tmp/c0130pis.pdf
How should Product C0130, Collagenase, be prepared for use in tissue digestions?
For use in tissue culture applications, Collagenase can be solubilized in calcium-free solutions such as Hanks Balanced Salts (H2387) or Earl's' Balanced Salt Solution (E6267).
What is the activity of the lot number of Product C0130, Collagenase, which I have?
The activity and specific activity for each lot number can be obtained from the certificate of analysis. To view the lot specific information click on the following link: http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=C0130|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC.   Under Useful Links & Tools, enter your lot number in the certificate of analysis field.
What type of Collagenases are present in Product C0130, Collagenase,?
This Collagenase is obtained from the culture filtrate of Clostridium histolyticum. The culture filtrate is thought to contain at least 7 different proteases ranging in molecular weight from 68 - 130 kDa. It does not contain any Matrix Metalloprotease.
Is Product C0130, Collagenase, sterile?
This product does not consist of a sterile preparation of Collagenase. Upon reconstitution, solutions should be sterile filtered through a 0.22 micron filter.
Which document(s) contains shelf-life or expiration date information for a given product?
If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis. These documents are located on the product detail page under Useful Links & Tools. Click on the following link to search for a Certificate of Analysis. Please click the following link to see the details on our Product Dating Information.
How do I get lot-specific information or a Certificate of Analysis?
A Certificate of Analysis is available by lot number and can be obtained through our Advanced Search Option: http://www.sigmaaldrich.com/catalog/AdvancedSearchPage.do
How do I find price and availability?
There are several ways to find pricing and availability for our products.  Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote.  USA customers:  1-800-325-3010 or view local office numbers. 
What is the Department of Transportation shipping information for this product?
Transportation information can be found in Section 14 of the product's (M)SDS. To access the shipping information for this material, use the link on the product detail page for the product, or search here. 
My question is not addressed here, how can I contact Technical Service for assistance?
Use the option to the right to "Ask a Question" by email of a Technical Service Scientist.
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Protocols & Articles

Protocols

Enzymatic Assay of Collagenase

The continuous spectrophotometric rate determination (A345, Light path = 1 cm) is based on the following reaction:
Keywords: Extinction coefficient

Related Content

Enzymes & Proteins

Application Index | Enzyme Index | Substrate Index | Inhibitor Index | Cofactor Index | Lectin Index
Keywords: Cell signaling, Diagnostic, Drug discovery, Molecular biology

Peer-Reviewed Papers
15

References

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inhibitor

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E6758 Ethylenediaminetetraacetic acid, anhydrous, crystalline, BioReagent, suitable for cell culture
E0396 Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioXtra, ≥97 .0%

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substrate

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C0627 Z-Gly-Pro-Leu-Gly-Pro

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