Read 26420310 Technical Tip Liberase Research Grade Enzyme Blends.indd text version

Liberase Research Grade Enzyme Blends

Technical Tip / April 2010

Calculation of working concentrations and conversion guide compared to traditional collagenases

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

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-fluorescent caseine assay (Matsubara, H. (1970)).

Liberase Research Grade Enzyme Liberase DL Research Grade Liberase DH Research Grade Liberase TL Research Grade Liberase TM Research Grade Liberase TH Research Grade Liberase Research Grade Selection Kit Enzyme Target Collagenase Target Neutral Target Mixture Protease Activity (Wünsch Collagenase Aggressiveness Amount units/vial) Content (mg/vial) 5 mg 50 mg 5 mg 50 mg 5 mg 5 mg 50 mg 5 mg 50 mg 5 mg of each enzyme 26 260 26 260 26 26 260 26 260 26 for each enzyme Low High Low Medium High See above + +++ ++ ++++ +++++ See above Neutral Protease Type Dispase Dispase Thermolysin Thermolysin Thermolysin See above

Endotoxin (EU/mg) 50 50 50 50 50 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... your application is included in our list* of applications your application is NOT included in our list of applications then... use the Liberase Research Grade concentration suggested in that application 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.roche-applied-science.com

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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 Liberase PI Liberase Blendzyme 1 Liberase CI Liberase RI Liberase Blendzyme 2 Liberase Blendzyme 3 Liberase Blendzyme 4 Liberase HI New Products (Liberase Research Grade Enzyme Blends) Liberase DL Research Grade Liberase DH Research Grade Liberase TL Research Grade* Liberase TM Research Grade** Liberase TH Research Grade 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.roche-applied-science/industrialbusiness

Previous Liberase Product Panel

Liberase Product PI RI HI CI Blendzyme 1 Blendzyme 2 Blendzyme 3 Blendzyme 4 Protein Mass (mg) 500 100 500 250 9 90 6 60 7 70 9 90 Target Collagenase (Wünsch units/vial) 2,250 470 2,325 1,000 28 280 28 280 28 280 28 280 Target Neutral Protease FITC (Casein units/vial) 55,000 20,000 100,000 49,000 3,000 30,000 6,000 60,000 12,000 120,000 24,000 240,000 Neutral Protease Type Dispase Thermolysin Thermolysin Thermolysin Dispase Thermolysin Thermolysin Thermolysin Neutral Protease/ Collagenase Unit Ratio 24 43 43 49 107 214 429 857

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.roche-applied-science.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 Purified 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.

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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 g/ml. Using the above Transition Guide, it 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 guidelines. 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 find its appropriate Liberase Research Grade Enzyme substitute.

Manufacturer Roche Roche Roche Roche Roche Manufacturer Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Type A B D P H Type I I II II III IV IV V V VII VII VII VII Specification crude crude crude crude crude Specification crude sterile crude sterile purified crude sterile crude sterile purified, culture tested purified purified, sterile purified, sterile, culture tested Wünsch Units/mg 0.15 0.15 0.15 1.50 0.15 Wünsch Units/mg 0.16 0.16 0.32 0.32 0.40 0.32 0.32 0.51 0.51 2.00 2.00 2.00 2.00 Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TH Research Grade Liberase TM Research Grade Liberase TL and DL Research Grade Liberase TM Research Grade Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TM Research Grade Liberase DH Research Grade Liberase DH Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TL Research Grade Liberase TL Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TM Research Grade

Technical Tip: Liberase Research Grade Enzyme Blends

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Manufacturer Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma

Type VIII XI XI XI XI IA IA IA IA F H L N

Specification crude crude culture tested sterile sterile, culture tested crude culture tested sterile sterile, culture tested blend blend blend blend

Wünsch Units/mg 0.32 0.90 0.90 0.90 0.90 0.32 0.32 0.32 0.32 0.51 0.33 0.18 0.03

Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TL Research Grade Liberase TL Research Grade Liberase TL Research Grade Liberase TL Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase DH Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TH Research Grade

Manufacturer Worthington Worthington Worthington Worthington Worthington Worthington Worthington Worthington Worthington Manufacturer Serva Serva Serva Manufacturer Gibco Gibco Gibco Gibco Gibco

Type 1 1 2 2 3 3 4 4 collagenase Type Coll. Hep + Coll. Pan + Collagenase Type Hepatocyte qualified I II III IV

Specification crude filtered crude filtered crude filtered crude filtered purified Specification crude crude crude Specification crude crude crude crude crude

Wünsch Units/mg 0.125 0.125 0.125 0.125 0.10 0.10 0.16 0.16 0.30 Wünsch Units/mg 0.70 0.70 0.70 Wünsch Units/mg 0.20 0.20 0.20 0.20 0.20

Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TM Research Grade Liberase TH Research Grade Liberase TH Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase DH Research Grade Liberase DH Research Grade Liberase TM Research Grade Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TL and DL Research Grade Liberase TM Research Grade Liberase Research Grade Enzyme Liberase TM Research Grade Liberase TH Research Grade Liberase TM Research Grade Liberase TM Research Grade Liberase TL and DL Research Grade

Technical Tip: Liberase Research Grade Enzyme Blends

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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 Purified 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 in your protocol, always express collagenase concentration in Wünsch units per milliliter of working enzyme solution.

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 final 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 Sigma Sigma, Serva, Worthington, Gibco To convert from FALGPA Units/mg CDU/mg or Mandl Units/mg To Wünsch Units/mg Wünsch Units/mg Divide FALGPA Units/mg by 3.9 CDU/mg by 1000 Example 3.5 FALGPA units/mg/3.9 = 0.9 Wunsch units/mg 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.

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Trademarks:

LIBERASE is a trademark of Roche. Dispase is a registered trademark of Godo Shusei Co., Ltd., Tokyo, Japan.

Intended Use:

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

Contact and Support

If you have questions or experience problems with this or any Roche Applied Science (RAS) product, please contact our Technical Support staff. Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing RAS product performance for using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to RAS and the world-wide research community. To ask questions, solve problems, suggest enhancements or report new appli-cations, please visit our Online Technical Support Site at: www.roche-applied-science.com/support To call, write, fax, or email us, visit the Roche Applied Science home page, www.roche-applied-science.com, and select your home country. Country-specific contact information will be displayed. On the Roche Applied Science home page select Printed Materials to find: in-depth Technical Manuals Lab FAQS: Protocols and references for life science research our quarterly Biochemica Newsletter Material Safety Data Sheets Pack Inserts and Product Instructions or to request hard copies of printed materials.

Published by: Roche Diagnostics GmbH Roche Applied Science Werk Penzberg 82372 Penzberg Germany © 2010 Roche Diagnostics GmbH All rights reserved.

Technical Tip: Liberase Research Grade Enzyme Blends

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