Calculation of working concentrations and conversion guide compared to traditional collagenases

Target Activities

Each vial of Liberase Research Grade Purified Enzyme Blend is filled based on total protein mass. Combined collagenolytic activity of the collagenase I and II isoforms is measured using the Wünsch method (Wünsch, E. & Heidrich, H.G. (1963)). Neutral protease activity is measured by a non-fl uorescent caseine assay (Matsubara, H. (1970)).

 

Target Collagenase Content (mg/vial) Liberase Research Grade Enzyme Target CollagenasT Activity (Wünsch units/vial) Target Neutral Protease Amount Enzyme Mixture Aggressiveness Neutral Protease Type Endotoxin (EU/mg)
Liberase DL

Research Grade
5 mg

50 mg
26

260
Low  +   Dispase   ≤ 50
Liberase DH

Research Grade
5 mg

50 mg
26

260
High  +++   Dispase   ≤ 50
Liberase TL 5 mg 26 Low  ++   Thermolysin   ≤ 50
Liberase TM

Research Grade
5 mg

50 mg
26

260
Medium  ++++   Thermolysin   ≤ 50
Liberase TH

Research Grade
5 mg

50 mg
26

260
High  +++++   Thermolysin   ≤ 50
Liberase

Research Grade

Selection Kit
5 mg of each enzyme 26 for each enzyme See above See above See above  ≤ 50 each

+ = lowest neutral protease activity/mg protein
+++++ = highest neutral protease activity/mg protein

Liberase Research Grade Enzyme Working Concentration

All Liberase Research Grade Enzymes have substantially higher specific activities than traditional collagenases. Therefore, the working concentration of Liberase Research Grade Purified Enzymes, expressed in mg/ml, will be much lower than that of traditional collagenase.

If... then...
your application is included in our list* of applications use the Liberase Research Grade concentration suggested in that application
your application is NOT included in our list of applications use Liberase TM Research Grade Enzyme at a concentration of 0.08 — 0.28 Wünsch units/ml

* Please find this list by visiting the Tissue Dissociation Special Interest Site at www.lifescience.roche.com

Exchange previous Liberases to Liberase Research Grade Enzyme Blends

Liberase enzyme blends of the 1st generation have been used successfully in many different applications.

Please refer to the following Transition Guide to substitute previous Liberase reagents with the appropriate new Liberase Research Grade product.

Transition Guide

Previous Liberase Products New Products (Liberase Research Grade Enzyme Blends)
Liberase PI Liberase DH Research Grade
Liberase Blendzyme 1 Liberase DH Research Grade
Liberase CI Liberase RI Liberase TL Research Grade*
Liberase Blendzyme 2 Liberase Blendzyme 3 Liberase TM Research Grade**
Liberase Blendzyme 4 Liberase TH Research Grade
Liberase HI Liberase Grade MTF C/T GMP***

*     composition corresponds to the former Liberase RI blend
**   composition corresponds to the former Liberase Blendzyme 3
*** For more information on the GMP-grade preparations, contact us at: www.lifescience.roche.com

 

 

Previous Liberase Product Panel

Liberase Product Protein Mass (mg) Target Collagenas Activity (Wünsch units/vial) Target Neutral Protease FITC (Casein units/vial) Neutral Protease Type Neutral Protease/Collagenase Unit Ratio
PI 500 2,250 55,000  Dispase  24
RI 100 470 20,000 Thermolysin 43
HI 500 2325 100,000 Thermolysin 43
CI 250 1,000 49,000 Thermolysin 49
Blendzyme 1 9 90 28 280 3,000 30,000 Dispase 107
Blendzyme 2 6 60 28 280 6,000 60,000 Thermolysin 214
Blendzyme 3 7 70 28 280 12,000 120,000 Thermolysin 429
Blendzyme 4 9 90 28 280 24,000 240,000 Thermolysin 857

Conversion from 1st generation Liberase and Liberase Blendzymes:

Follow the protocols for the 1st generation Liberase products, and simply adjust by using the new concentration indicated above.

Liberase enzyme blends of the 1st generation have been used successfully in the applications listed on the Tissue Dissociation
Special Interest Site at www.lifescience.roche.com.

Select the appropriate protocol and calculate the working concentration of the new 2nd generation Liberase Research Grade Enzyme as follows:

The mg/vial amounts given for the 1st generation Liberase and Liberase Blendzymes refers to whole protein amounts (collagenase I + collagenase II + neutral protease). In contrast, the mg/vial indications of the 2nd generation Liberase Research Grade Puri ed Enzyme Blends refer to total collagenase amounts, without considering the protein amount of neutral proteases.

For this reason, it is essential to express collagenase concentration of the 1st generation Liberase Enzymes in Wünsch units/ml, instead of mg/ml.

The Wünsch units/ml of 1st and 2nd generation products are equivalent.

Example

Let's assume you have selected the protocol for isolating human hepatocytes from the table, which recommends using the 1st generation Liberase CI in a final concentration of 100 μg/ml. Using the above Transition Guide, it is possible to the 1st generation Liberase CI in a nal concentration of 100 μg/ml. Using the above Transition Guide, is possible to identify Liberase TL Research Grade as the 2nd generation successor to the former Liberase CI.

To obtain the new starting concentration of 2nd generation Liberase TL Research Grade, use the collagenase concentration of the first generation product in Wunsch units/ml, instead of mg/ml.

Liberase CI: 250 mg = 1000 Wünsch units, working concentration: 100 µg/ml = 0.4 Wünsch units/ml.
Liberase TL Research Grade: 2.5 mg = 13 Wünsch units, working concentration: 0.4 Wünsch units/ml = 77 µg/ml.
To isolate human hepatocytes, start with a concentration of 80 µg/ml of Liberase TL Research Grade Enzyme.

Note: Second generation Liberase Research Grade Enzymes have significantly higher purity collagenase I + II in comparison to the older Liberase enzymes. Therefore, new incubation time will have to be determined for each application. In most applications, a 10-30% reduction in incubation time will yield optimal results.

Substitution for other Collagenases

Despite the availability of Liberase Research Grade Enzyme Blends, tissue dissociation is an inexact science. Different methods use different enzyme types and concentrations, even when using the same tissue dissociation protocol for the same tissue and species. Concentration information provided in this Technical Tip are starting . Each protocol may require additional enzyme optimization, depending on your experimental goals. Once you have found the appropriate Liberase Research Grade Enzyme type and concentration for your protocol, it should not be necessary adapt to different lots due to a high lot-to-lot consistency of the Liberase products.

Collagenase Enzyme Conversion Table

Find your traditional collagenase enzyme in the table below, and then fi nd its appropriate Liberase Research Grade Enzyme substitute.

Manufacturer Type Specification Wünsch Units/mg Liberase Research Grade Enzyme
Roche A crude 0.15 Liberase TM Research Grade
Roche B crude 0.15 Liberase TM Research Grade
Roche D crude 0.15 Liberase TM Research Grade
Roche P crude 1.5 Liberase TL and DL Research Grade
Roche H crude 0.15 Liberase TM Research Grade

 

Manufacturer Type Specification Wünsch Units/mg Liberase Research Grade Enzyme
Sigma I crude 0.16 Liberase TM Research Grade
Sigma I sterile 0.15 Liberase TM Research Grade
Sigma II crude 0.32 Liberase DH Research Grade
Sigma II sterile 0.32 Liberase DH Research Grade
Sigma III purified 0.40 Liberase TM Research Grade
Sigma IV crude 0.32 Liberase TM Research Grade
Sigma IV sterile 0.32 Liberase TM Research Grade
Sigma V crude 0.15 Liberase TL Research Grade
Sigma V sterile 0.51 Liberase TL Research Grade
Sigma VII purified, culture tested 2.00 Liberase TM Research Grade
Sigma VII purified 2.00 Liberase TM Research Grade
Sigma VII purified, sterile 2.00 Liberase TM Research Grade
Sigma VII purified, sterile, culture tested 2.00 Liberase TM Research Grade
Sigma VIII crude 0.32 Liberase TM Research Grade
Sigma XI crude 0.90 Liberase TL Research Grade
Sigma XI culture tested 0.90 Liberase TL Research Grade
Sigma XI sterile 0.15 Liberase TL Research Grade
Sigma XI sterile, culture tested 0.90 Liberase TL Research Grade
Sigma IA crude 0.32 Liberase TM Research Grade
Sigma IA culture tested 0.32 Liberase TM Research Grade
Sigma IA sterile 0.32 Liberase TM Research Grade
Sigma IA sterile, culture tested 0.32 Liberase TM Research Grade
Sigma F blend 0.51 Liberase DH Research Grade
Sigma H blend 0.33 Liberase TM Research Grade
Sigma L blend 0.18 Liberase TM Research Grade
Sigma N blend 0.03 Liberase TH Research Grade

 

Manufacturer Type Specification Wünsch Units/mg Liberase Research Grade Enzyme
Worthington 1 crude 0.125 Liberase TM Research Grade
Worthington 1 filtered 0.125 Liberase TM Research Grade
Worthington 2 crude 0.125 Liberase TH Research Grade
Worthington 2 filtered 0.125 Liberase TH Research Grade
Worthington 3 crude 0.10 Liberase TM Research Grade
Worthington 3 filtered 0.10 Liberase TM Research Grade
Worthington 4 crude 0.16 Liberase DH Research Grade
Worthington 4 filtered 0.16 Liberase DH Research Grade
Worthington collagenase purified 0.30 Liberase TM Research Grade

 

Manufacturer Type Specification Wünsch Units/mg Liberase Research Grade Enzyme
Serva Coll. Hep + crude 0.70 Liberase TM Research Grade
Serva Coll. Pan + crude 0.70 Liberase TL and DL Research Grade
Serva Collagenase crude 0.70 Liberase TM Research Grade

 

Manufacturer Type Specification Wünsch Units/mg Liberase Research Grade Enzyme
Gibco Hepatocyte qualified crude 0.20 Liberase TM Research Grade
Gibco I crude 0.20 Liberase TH Research Grade
Gibco II crude 0.20 Liberase TM Research Grade
Gibco III crude 0.20 Liberase TM Research Grade
Gibco IV crude 0.20 Liberase TL and DL Research Grade

Conversion from traditional Collagenases:

Conversion from traditional Collagenases:
Follow the protocols for the traditional collagenases and adjust to the new concentration.

Calculate the working concentration of the new 2nd generation Liberase Research Grade Enzyme as follows:

The mg/vial amounts given for the 1st generation Liberase and traditional collagenases refer also generally to whole protein amounts (collagenase + neutral protease). To substitute using 2nd generation Liberase Research Grade Purifi ed Enzyme, it is important to first convert the concentration in Wünsch units/ml, instead of mg/ml.

Multiply your previous collagenase working concentration (mg/ml) by its specific activity (Wünsch units/mg) to obtain Wünsch units/ml. For consistency

Example:
Using the Transition Guide, it is possible toidentify Liberase TL/DL Research Grade as the 2nd generation successor to Collagenase P from Roche.

To obtain the new concentration of 2nd generation Liberase TL/DL Research Grade, calculate the working concentration in Wünsch units/ml using the given Wünsch units/mg in the tables above of Collagenase P and use the same fi nal concentration of Liberase TL/DL Research Grade in Wünsch units/ml. Collagenase P: 1.5 Wünsch units/mg, working concentration: 0.5 – 1.5 mg/ml
= 0.75 – 2.25 Wünsch units/ml Liberase TL/DL Research Grade: use 0.75 – 2.25 Wünsch units/ml

Example calculation for an application:
For the isolation of rat hepatocytes 1 mg/ml Collagenase H is used.

To obtain the new starting concentration of 2nd generation Liberase TM Research Grade, calculate the working concentration in Wünsch units/ml and use the same concentration of Wünsch units/ml.

Collagenase H: 0.15 Wünsch units/mg, working concentration: 1 mg/ml = 0.15 Wünsch units/ml. Liberase TM Research Grade: use 0.15 Wünsch units/ml.

Convert Collagenase specific activity to Wünsch U/mg

Use the following table to convert collagenase enzyme activity of your current collagenase to Wünsch (collagenase) units/mg. This table calculates Wünsch units/mg from either FALGPA units/mg or collagen degrading units/mg (Mandl units; CDU).

Supplier To convert from To Divide Example
Sigma FALGPA Units/mg Wünsch Units/mg FALGPA Units/mg by 3.9 3.5 FALGPA units/mg/3.9 = 0.9 Wunsch units/mg
Sigma, Serva, Worthington, Gibco CDU/mg or Mandl Units/mg Wünsch Units/mg CDU/mg by 1000 200 CDU/mg/1000 = 0.2 Wünsch units/mg
Note: These conversions are a reasonable approximation taking into account the expected accuracy of the different collagenase assays.

Visit our Liberase Enzyme website at www.collagenase.com for information how to optimize your tissue dissociation procedure.

Trademarks

LIBERASE is a trademark of Roche.
Dispase is a registered trademark of Godo Shusei Co., Ltd.,y Too, kJapan.

Intended Use

For life science research only. Not for use in diagnostic procedures.