For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.

Chromozym t-PA

1. What this Product Does

ApplicationChromozym t-PA is used as substrate for the determination of t-PA,both in purified preparations and in cell culture supernatants. More-over, it can be applied to evaluate the content of one-chain and two-chain t-PA.

Storage and StabilityThe preparation is stable at +15 to +25°C through the expiration dateprinted on the label.

Product Characteristics

Reaction PrincipleChromozym t-PA is cleaved by t-PA under formation of 4-nitraniline,which is measured at 405 nm. The t-PA activity is calculated in U/mlfrom the absorbance difference per min (see procedure 1). How-ever, it has to be considered that one chain t-PA reacts lesssensitive with oligopeptide substrated than two chain t-PA (1). t-PAin the sample can be measured in defined two chain form afterincubation with plasmin (2) (see procedure 2):CH3-SO2-D-Phe-Gly-Arg-4-nitranilide+ H2O → CH3-SO2-D-Phe-Gly-

Arg-OH + 4-nitraniline

Additional Reagents Required(for procedure 1)• Tris* [2-amino-2-(hydroxymethyl)-1,3-propane diole]• 1 M Hydrochloric acid• Tween 80 (polyoxyethylenesorbitane monooleate)• Citric acid-1-hydrate, A.R.l(additionally for procedure 2)• Plasmin from human plasma*• Aprotinin*

2. How to Use this Product

2.1 Before You Begin

Preparation of Additional Solutions Required(for procedure 1)

Sample Materialt-PA solutions in the concentration range of 0.1-30 �g/ml. Higherconcentrated t-PA solutions should be diluted with Tris buffer (1).

N-Methylsulfonyl-D-Phe-Gly-Pro-Arg-4-nitranilide acetate

Cat. No. 11 093 037 001 20 mg Version June 2010Store at +15 to +25°C

Molecular Weight 636.7

Structure N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranil-ide-acetate

Formula C24H32N8O7S × CH3COOH

Form of Supply Powder

Purity 90% N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranilide acetate (enzymatic)

Contaminants <0.5% free 4-nitraniline

Working Concentration

approx. 0.25 mM

Solution Composition/Preparation Storage/ Stability

for procedure 1

1 Tris buffer (100 mM Tris, pH 8.5, Tween 80, 0.15%, w/v)

Dissolve 121 mg Tris with redist. water. Adjust to pH 8.5 with 1 M HCl. Sepa-rately dissolve 0.15 g Tween 80 with 10 ml redist water and add to the Tris solution. Fill up to 100 ml with redist. water.

Stable for at least 2 weeks if stored at +2 to +8°C.

2 Chromozym t-PA solu-tion, 4 mM

Dissolve 5.1 mg Chro-mozym t-PA with 2 ml redist. water.

Stable for at least 2 weeks if stored at +2 to +8°C.

3 Citric acid solution, 10 % (w/v)

Dissolve 10.94 g citric acid in 100 ml double-distilled water.

Stable for at least 4 weeks if stored at +2 to +8°C.

4 Reagent mixture

Mix 9 parts Tris buffer (1) with 1 part Chromozym t-PA solution (2).

Stable for 4 h at +15 to +25°C and fot 8 h at +2 to +8°C.

Additionally required for procedure 2

5 Plasmin solution, 8 U/ml

Dissolve 4 U plasmin with 0.5 ml Tris buffer (1). The plasmin activity is deter-mined at +25°C with Chro-mozym PL as substrate.

Stable in an ice-bath until starting the assay, can be frozen in aliquots.

6 Aprotinin solution, 100 �g/ml

Dissolve 5 mg aprotinin with 50 ml redist. water.

Stable for approx. 1 week at +4°C, can be frozen.

www.roche-applied-science.com0610.110955280016

2.2 Procedure 1Apply the following photospectrometer parameters:• Wave length: Hg 405 nm• Plastic cuvettes• Light path: 1 cm• Temperature: 37°C• Assay volume: 1.60 ml• Measure against reagent blank.• Bring reagent mixture (solution 4) to 37°C before using the assay.• At least one reagent blank should be run per each measuring

series.

2.3 Calculation of the t-PA activity in International Units (U/ml)

If diluted samples are assayed the dilution factor F has to be taken intoaccount:

2.4 Calculation of the t-PA activity WHO Units (IU/ml)For calculation in WHO Units a t-PA standard has to be run in parallel.

L One International Unit (U) t-PA � approx. 32,500 WHO Units (IU)t-PA.

2.5 Procedure 2Apply the following photospectrometer parameters:• Wave length: Hg 405 nm• plastic cuvettes• Light path: 1 cm• Temperature: 37°C• Assay volume: 1.75 ml• Measure against reagent blank.• Bring Tris buffer (solution 1) and Chromozym t-PA solution (solu-

tion 2) to 37°C before using the assay.• At least one reagent blank should be run per each measuring

series.

2.6 Calculation of the t-PA activity in International Units (U/ml)

If diluted samples are assayed, the dilution factor F has to be takeninto account:

For calculation of the t-PA activity in WHO Units see assay procedure1.N Use only plastic materials (no glass!) for storage, dilution and the

assay of t-PA.L Concerning the determination of t-PA using plasminogen refer to

the literature (2).L Concerning the determination of one- and two-chain t-PA in the

same sample material with oligopeptide substrates some methodsare described in the literature (3).

Step Action

t-PA sampleMeasuring Range Reagent

Blank0.1 - 2.5 �g/ml

1 - 30 �g/ml

� Into a plastic cuvette pipet:

t-PA sample 0.1 ml 0.1 ml �

Tris buffer (solution 1) — � 0.1 ml

Reagent mixture (solution 2)

1.0 ml 1.0 ml 1.0 ml

� Mix and incubate at +37°C for

30 min 3 min 3 or 30 min

� Add citric acid solution (3)

0.5 ml 0.5 ml 0.5 ml

� Mix and read adsorbance of the sample against reagent blank (= �Asample).

t-PA (U/ml) = �Asample/min × 1.6

10.4 × 0.1

t-PA (U/ml) = �Asample/min × 1.54

t-PA (U/ml) = �Asample/min × 1.54 � F

t-PA (U/ml) = �Asample × IU/mlstandard

�Asample

Step Action

t-PA sampleMeasuring Range Reagent

Blank0.1 - 2.5 �g/ml

1 - 30 �g/ml

� Into a plastic cuvette pipet:

Tris buffer (solution 1)

1.0 ml 1.0 ml 1.1 ml

t-PA sample 0.1 ml 0.1 ml �

Plasmin solution (solution 5)

0.025 ml 0.025 ml 0.025 ml

� Mix and incubate at 37°C for 5 min.

� Add Aprotinin solu-tion (solution 6)

0.025 ml 0.025 ml 0.025 ml

� Mix and incubate at 37°C for 5 min.

� Add Chromozym t-PA solution (solution 2)

0.1 ml 0.1 ml 0.1 ml

� Mix and incubate at 37°C for

30 min 3 min 3 or 30 min

� Add citric acid solu-tion (solution 3)

0.5 ml 0.5 ml 0.5 ml

� Mix and read adsorbance of the sample against reagent blank (= �Asample).

t-PA (U/ml) = �Asample/min × 1.75

10.4 × 0.1

t-PA (U/ml) = �Asample/min × 1.68

t-PA (U/ml) = �Asample/min × 1.68 � F

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2.7 References1 Ranby, M., Bergsdorf, N. & Nilsson, T. (1982) Thromb. Res. 27,

175-183.2 Lill, H. (1987) Z. gesamte inn. Med. 42, 478-486.3 Verheijen, J. H., de Jong, Y. F. & Chang, G. T. G. (1985) Thromb.

Res. 39, 281-288.

3. Supplementary Information

3.1 Conventions

Text ConventionsTo make information consistent and memorable, the following textconventions are used in this package insert:

SymbolsIn this Instruction Manual, the following symbols are used to highlight impor-tant information:

3.2 Ordering InformationRoche Applied Science offers a large selection of reagents and systems for lifescience research. For a complete overview of related products and manuals,please visit and bookmark our home page, www.roche-applied-science.com.

3.3 TrademarksCHROMOZYM is a registered trademark of Pentapharm AG, Basle, SwitzerlandTWEEN is a registered trademark of ICI Americas Inc., Wilmington USA

Regulatory DisclaimerFor life science research only. Not for use in diagnostic procedures.

Symbol Description

Numbered stages labeled , , etc.

Stages in a process that usually occur in the order listed.

Numbered Instructionslabeled �, �, etc.

Steps in a procedure that must be per-formed in the order listed

Asterisk * Denotes a product available from Roche Applied Science

Symbol Description

L Information Note:Additional information about the current topic or procedure.

N Important Note:Information critical to the success of the procedure or use of the product.

Product Pack Size Cat No.

Chromozym PK 20 mg 10 378 445 001

Chromozym TH 20 mg100 mg

10 206 849 00111 585 398 001

Plasmin, human 5 U 10 602 361 001

Tris base 500 g1 kg5 kg

10 708 968 00110 708 976 00111 814 273 001

Aprotinin (from bovine lung 10 gm50 mg100 mg

10 236 624 00110 981 532 00111 583 794 001

Germany