ThermoGMP

Animal Free Thermolysin

$160.00

Description

Animal-Free (AF) Thermolysin is an aseptically dispensed purified protease from Bacillus thermoproteolyticus supplied as 6 mg of lyophilized powder.

Intended Use

AF Thermolysin is a thermostable neutral protease that can be used with purified collagenase to isolate many different types of cells. It is commonly used with collagenase to isolate islets from rodent, cynomolgus monkey¹, and human pancreata.

Cat. # 002-3000
  • GMP Grade enzyme suitable for ex-vivo use in isolating cells for a variety of applications
  • Manufactured for superior lot-to-lot enzyme consistency for reliable performance
  • Low contaminating endotoxin levels (<50 EU/mg)
  • Lot specific Certificate of Analysis provided with complete QC analysis results
  • White lyophilized cake under vacuum in amber bottle sealed with butyl rubber stopper
  • Contains >2.3 Million Neutral Protease Activity (NPA) units.
  • Unopened product stable for at least 2 years when stored between -15 to – 25°C; internal studies indicate the reconstituted enzyme is stable at the same temperatures for at least 1 year, as long as no other protease enzyme has been added to the solution.
  • Product is shipped on dry ice to provide the most stable conditions during shipment.

Manufacturing Details

AF Thermolysin is purified from B. thermoproteolyticus animal origin free culture supernatants and purified by re-crystallization and column chromatography. After assessing enzyme potency, the AF is sterile filtered and aseptically dispensed into depyrogenated vials and lyophilized. When dry, the AF Thermolysin vials are sealed under vacuum. Representative samples from the batch are reconstituted and quality control tested. Results are compared to established specifications before release into inventory.

Suggested Assays

The working concentration of purified thermolysin is determined by the total enzyme activity contained in the product. Typically, this is expressed as the total neutral protease units as measured by using a FITC-BSA substrate. Several reports have shown the importance of using an optimal amount of neutral protease in an islet isolation procedure.

References

Product Documentation

Pack Insert

Safety Data Sheet

Specifications

Certificate of Origin

General Product FAQ

Are your enzymes animal-free?

Thermolysin and BP Protease are entirely animal origin free.

A porcine gelatin peptone is used during the fermentation step of natural collagenase production. While this animal sourced material is required to stimulate collagenase production from the biosynthesis from the organism Clostridium histolyticum during anaerobic fermentation, it represents a very low adventitious agent risk as the media is sterilized at 121°C for 30 minutes prior to inoculation, which has been shown to inactivate many viruses.

The gelatin is removed during the purification process and no animal sourced materials are used in the remainder of the process.

Additional manufacturing details are available in product insert literature for each product.

What are application-specific formulations?

Application-specific formulations are pre-blended mixtures of purified collagenase and protease in optimal ratios for isolation of cells from a particular tissue and/or species.

Currently, our CIzyme™ AS and CIzyme™ RI are our only two application-specific formulations.

What is the best way to inactivate the enzymes when a digestion is complete?

There are two components to inactivation of the digestive enzymes commonly used for tissue dissociation.  To inactivate protease activity, a serum-containing media or buffer is most effective.  Buffers or media containing human serum albumin (HSA) are ineffective in suppressing protease activity, even at very high concentrations of protein (>10%). However, serum-containing buffers do not inhibit collagenase activity.  The most effective method for inactivating collagenase activity is reducing temperature. Collagenases will lose about 80% their maximal activity at 26°C. Thus, introduction of an ice-cold, serum- containing media should virtually eliminate all collagenolytic and proteolytic activity of an enzyme mixture.

General Protease FAQ

Can I substitute the type of protease I use for my application?

Yes.  Selection of a protease in many cases is simply due to the historical precedence of the given cell isolation protocol being followed; however, that is not to say a different one would not work.  The isolation of human islets is a perfect example in which thermolysin, BP Protease (a Dispase equivalent), and C. histolyticum neutral protease have all been successfully used.  Generally, bacterial or fungal sources of protease are selected due to their broader specificity. Substitution of one protease for another may not be a seamless transition, however, as the kinetics of digestion can be dramatically different among the various general proteases.   For guidance in selecting a protease, contact VitaCyte’s technical support team.

Thermolysin FAQ

How should I store my AF Thermolysin?

This product is stable for at least two years from date of manufacture if stored unopened between -15 to – 25°C. Internal studies have shown the reconstituted enzyme is stable as a frozen solution between -15 to – 25°C for at least 1 year. Additional studies have shown the enzyme can go through a freeze-thaw cycle at least once without loss of potency by neutral protease (NP) assay. The product is shipped on dry ice to provide the most stable conditions during shipment.