For life science research only.

Not for use in

diagnostic procedures. FOR IN VITRO USE ONLY.

Chromozym t-PA
N-Methylsulfonyl-D-Phe-Gly-Pro-Arg-4-nitranilide acetate

Cat. No. 11 093 037 001 20 mg Version June 2010

Store at +15 to +25C

1. What this Product Does 2. How to Use this Product

Application 2.1 Before You Begin
Chromozym t-PA is used as substrate for the determination of t-PA,
both in purified preparations and in cell culture supernatants. More- Preparation of Additional Solutions Required
over, it can be applied to evaluate the content of one-chain and two- (for procedure 1)
chain t-PA.
Solution Composition/Preparation Storage/ Stability
Storage and Stability
for procedure 1
The preparation is stable at +15 to +25C through the expiration date
printed on the label. 1 Tris buffer Dissolve 121 mg Tris with Stable for at least 2
(100 mM redist. water. Adjust to pH weeks if stored at
Product Characteristics Tris, pH 8.5, 8.5 with 1 M HCl. Sepa- +2 to +8C.
Tween 80, rately dissolve 0.15 g Tween
0.15%, 80 with 10 ml redist water
Reaction Principle w/v) and add to the Tris solution.
Chromozym t-PA is cleaved by t-PA under formation of 4-nitraniline, Fill up to 100 ml with redist.
which is measured at 405 nm. The t-PA activity is calculated in U/ml water.
from the absorbance difference per min (see procedure 1). How-
2 Chromozym Dissolve 5.1 mg Chro- Stable for at least 2
ever, it has to be considered that one chain t-PA reacts less
sensitive with oligopeptide substrated than two chain t-PA (1). t-PA t-PA solu- mozym t-PA with 2 ml weeks if stored at
in the sample can be measured in defined two chain form after tion, 4 mM redist. water. +2 to +8C.
incubation with plasmin (2) (see procedure 2): 3 Citric acid Dissolve 10.94 g citric acid Stable for at least
CH3-SO2-D-Phe-Gly-Arg-4-nitranilide+ H2O CH3-SO2-D-Phe-Gly- solution, in 100 ml double-distilled 4 weeks if stored
Arg-OH + 4-nitraniline 10 % (w/v) water. at +2 to +8C.
4 Reagent Mix 9 parts Tris buffer (1) Stable for 4 h at
Molecular Weight 636.7 mixture with 1 part Chromozym t- +15 to +25C and
PA solution (2). fot 8 h at +2 to
Structure N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranil- +8C.
ide-acetate
Additionally required for procedure 2
Formula C24H32N8O7S CH3COOH
5 Plasmin Dissolve 4 U plasmin with Stable in an ice-
Form of Supply Powder solution, 0.5 ml Tris buffer (1). The bath until starting
Purity 90% N-Methylsulfonyl-D-Phe-Gly-Arg-4- 8 U/ml plasmin activity is deter- the assay, can be
nitranilide acetate (enzymatic) mined at +25C with Chro- frozen in aliquots.
mozym PL as substrate.
Contaminants <0.5% free 4-nitraniline
6 Aprotinin Dissolve 5 mg aprotinin Stable for approx.
Working approx. 0.25 mM solution, with 50 ml redist. water. 1 week at +4C,
Concentration 100 g/ml can be frozen.
Additional Reagents Required Sample Material
(for procedure 1) t-PA solutions in the concentration range of 0.1-30 g/ml. Higher
Tris* [2-amino-2-(hydroxymethyl)-1,3-propane diole] concentrated t-PA solutions should be diluted with Tris buffer (1).
1 M Hydrochloric acid
Tween 80 (polyoxyethylenesorbitane monooleate)
Citric acid-1-hydrate, A.R.l
(additionally for procedure 2)
Plasmin from human plasma*
Aprotinin*

0610.110955280016 www.roche-applied-science.com
2.2 Procedure 1 2.5 Procedure 2
Apply the following photospectrometer parameters: Apply the following photospectrometer parameters:
Wave length: Hg 405 nm Wave length: Hg 405 nm
Plastic cuvettes plastic cuvettes
Light path: 1 cm Light path: 1 cm
Temperature: 37C Temperature: 37C
Assay volume: 1.60 ml Assay volume: 1.75 ml
Measure against reagent blank. Measure against reagent blank.
Bring reagent mixture (solution 4) to 37C before using the assay. Bring Tris buffer (solution 1) and Chromozym t-PA solution (solu-
At least one reagent blank should be run per each measuring tion 2) to 37C before using the assay.
series. At least one reagent blank should be run per each measuring
series.
Step Action
t-PA sample Step Action
Measuring Range Reagent t-PA sample
0.1 - 2.5 1 - 30 Blank Measuring Range Reagent
g/ml g/ml 0.1 - 2.5 1 - 30 Blank
Into a plastic cuvette pipet: g/ml g/ml
t-PA sample 0.1 ml 0.1 ml Into a plastic cuvette pipet:
Tris buffer (solution 1) 0.1 ml Tris buffer 1.0 ml 1.0 ml 1.1 ml
(solution 1)
Reagent mixture 1.0 ml 1.0 ml 1.0 ml
(solution 2) t-PA sample 0.1 ml 0.1 ml
Mix and incubate at 30 min 3 min 3 or 30 min Plasmin solution 0.025 ml 0.025 ml 0.025 ml
+37C for (solution 5)
Add citric acid solution 0.5 ml 0.5 ml 0.5 ml Mix and incubate at 37C for 5 min.
(3) Add Aprotinin solu- 0.025 ml 0.025 ml 0.025 ml
Mix and read adsorbance of the sample against reagent blank tion (solution 6)
(= Asample). Mix and incubate at 37C for 5 min.
Add Chromozym 0.1 ml 0.1 ml 0.1 ml
2.3 Calculation of the t-PA activity in International Units
(U/ml) t-PA solution
(solution 2)
Asample/min 1.6 Mix and incubate at 30 min 3 min 3 or 30 min
t-PA (U/ml) = 37C for
10.4 0.1
Add citric acid solu- 0.5 ml 0.5 ml 0.5 ml
tion (solution 3)
t-PA (U/ml) = Asample/min 1.54
Mix and read adsorbance of the sample against reagent blank
If diluted samples are assayed the dilution factor F has to be taken into (= Asample).
account:
2.6 Calculation of the t-PA activity in International Units
t-PA (U/ml) = Asample/min 1.54 F (U/ml)

2.4 Calculation of the t-PA activity WHO Units (IU/ml) Asample/min 1.75
For calculation in WHO Units a t-PA standard has to be run in parallel. t-PA (U/ml) =
10.4 0.1
Asample IU/mlstandard
t-PA (U/ml) = t-PA (U/ml) = Asample/min 1.68
Asample
L One International Unit (U) t-PA approx. 32,500 WHO Units (IU)
If diluted samples are assayed, the dilution factor F has to be taken
t-PA. into account:

t-PA (U/ml) = Asample/min 1.68 F

For calculation of the t-PA activity in WHO Units see assay procedure
1.
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 methods
are described in the literature (3).

2
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2.7 References
1 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 Conventions
To make information consistent and memorable, the following text
conventions are used in this package insert:

Symbol Description
Numbered stages Stages in a process that usually occur in
labeled , , etc. the order listed.
Numbered Instructions Steps in a procedure that must be per-
labeled , , etc. formed in the order listed
Asterisk * Denotes a product available from Roche
Applied Science

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

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.

3.2 Ordering Information

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

Product Pack Size Cat No.

Chromozym PK 20 mg 10 378 445 001
Chromozym TH 20 mg 10 206 849 001
100 mg 11 585 398 001
Plasmin, human 5U 10 602 361 001
Tris base 500 g 10 708 968 001
1 kg 10 708 976 001
5 kg 11 814 273 001
Aprotinin (from bovine lung 10 gm 10 236 624 001
50 mg 10 981 532 001
100 mg 11 583 794 001

3.3 Trademarks
CHROMOZYM is a registered trademark of Pentapharm AG, Basle, Switzerland
TWEEN is a registered trademark of ICI Americas Inc., Wilmington USA

Regulatory Disclaimer
For life science research only. Not for use in diagnostic procedures.
Contact and Support
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