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QuantiChromTM Arginase Assay Kit (DARG-200)
Quantitative Colorimetric Arginase Determination
DESCRIPTION
ARGINASE (L-arginine ureohydrolase EC 3.5.3.1) is
present in
mammals and plants. In
humans, arginase is expressed predominantly in the liver, and to lesser
degrees in breast, kidney, testes, salivary glands, epidermis and
erythrocytes. Arginase catalyzes the conversion of arginine to ornithine
and urea, completing the last step in the urea cycle. Arginase activity
is a key diagnostic indicator. Increased levels of arginase activity in
blood have been associated with liver damage [1]. Hyperargininemia due
to arginase deficiency is an inherited autosomal recessive disease
[2].Simple, direct and automation-ready procedures for measuring
arginase activity in biological samples are highly desirable in Research
and Drug Discovery. BioAssay Systems' arginase assay kit provides a
sensitive and convenient method for arginase activity determination. The
method utilizes a chromogen that forms a colored complex specifically
with urea produced in the arginase reaction. The intensity of the color
is directly proportional to the arginase activity in the sample.
KEY FEATURES
Sensitive and accurate .
Detection limit: 1 U/L arginase activity in 96-well assay format.
Simple and high-throughput.
The procedure involves incubation of the provided substrate with the
sample in a microplate, addition of the coloring reagent and incubation
for 15 min. Can be readily automated as a high-throughput assay for
thousands of samples per day.
APPLICATIONS:
Direct Assays:
arginase activity in enzyme
preparations, serum, plasma, tissue culture etc;
Drug Discovery/Pharmacology: effects of drugs on
arginase activity.
KIT CONTENTS (for 200 samples in 96-well
assay)
Arginine Buffer (pH 9.5): 2 mL Mn Solution: 1 mL
Reagent A: 25 mL Reagent B: 25 mL
Urea standard: 1 mL 50mg/dL
Storage conditions .
Kit is shipped at room temperature. Store the Arginine Buffer and Urea
Standard at -20°C,
and other components at 2- 8°C. Shelf life:
at least 6 months (see expiry dates on labels).
Precautions :
reagents are for research use only. Normal precautions for laboratory
reagents should be exercised while using the reagents. Please refer to
Material Safety Data Sheet for detailed information.
PROCEDURES
Reagent Preparation: bring reagents to room
temperature prior to assay.
5x Substrate Buffer :
combine 4 vol of Arginine Buffer and 1 vol of the Mn Solution. For each
test, 10
μL 5x Substrate Buffer is needed.
Urea Reagent: combine equal volumes of Reagent A
and Reagent B.
1 mM Urea Standard: mix 24 μL 50mg urea /dL and
176 μL water.
Important: use the above reagents
within 2 hours after preparation.
Standard procedure using 96-well plate (200 tests):
1. Arginase Reaction: combine 40 μL sample and
10 μL 5x Substrate Buffer into wells of a clear bottom 96-well reaction
plate. In addition, transfer 40 μL sample without
5x Substrate Buffer (Sample
Blank Control, ODBLANK), 50
μL H2O (standard background, ODWATER) and 50 μL 1 mM Urea Standard
(ODSTANDARD) into separate wells of the reaction plate and incubate at
37°C for 2 hours.
Note:
samples may need to be
diluted with water depending on arginase activity. Assay works best if
sample is diluted so apparent activity lies between 1 and 40 U/L. Serum
or plasma samples contain urea which may need to be removed prior to
assaying (see General Considerations).
2.
Urea Determination: Add 200 μL Urea Reagent to all wells (note:
Urea Reagent stops arginase reaction)
and then add 10 μL 5x Substrate Buffer to the Sample Blank Control
well. Tap the
plate to mix.
3. Incubate 60 min at room temperature and read
optical density at 430nm or incubate 20 min for measurement at 520nm.
Note :
for some samples addition of urea reagent may cause turbidity. If this
occurs, transfer sample to an Eppendorf tube and centrifuge for 5
minutes at 14000 rpm. Transfer supernatant back to reaction plate and
read the absorbance.
CALCULATION
Arginase activity (units per liter of sample) is calculated as
OD SAMPLE,
ODBLANK,
ODSTANDARD
and ODWATER
are optical density values of sample, sample blank, standard and water,
respectively.
[Urea Standard] = 1 mM,
t is the reaction time (120 min). 50 and 40
are the reaction and sample volumes (μL),
respectively.
Unit definition: 1 unit of arginase converts 1
μmole of L-arginine to ornithine and urea per minute at pH 9.5 and 37°C.
GENERAL CONSIDERATIONS
A. The incubation time for the arginase reaction
(Step 1) can vary (0.5 to 4 hours) depending on the arginase activity.
If (ODSAMPLE – ODBLANK)/(ODSTANDARD – ODWATER) is larger than 3.5,
dilute sample in distilled water and repeat the assay, multiply the
results by the dilution factor.
B.
Sample Pretreatment: serum or plasma
samples contain urea. Urea can be depleted using a membrane filter (e.g.
Microcon YM-10 from Millipore). The recommended procedure,
1. Load up to 100
μL sample in a Microcon YM-10 (10 kDa cutoff) and
dilute with water to 500 μL.
Centrifuge at 14000 rpm for 30 min, check level of sample, ideally the
sample level will be less than 50 uL. Add water to 500 μL and repeat the
centrifugation.
2. Decant concentrated sample diluent and measure
final volume with a pipetman. Adjust final volume so there will be
enough sample for the reaction and reaction blan
C. Cell Lysate. ~106 cells per sample
are harvested, washed with PBS, and centrifuged at 1000g at 4°C
for 10 min. Pellets are lysed for 10 min in 100 μL
of 10 mM Tris-HCl (pH 7.4) containing 1
μM pepstatin A, 1 μM leupeptin, and 0.4% (w/v)
Triton X-100. Samples are centrifuged at 20,000g at 4°C for 10
min. Use supernatant for
arginase assay.
MATERIALS REQUIRED, BUT NOT PROVIDED
Pipetting devices and accessories, clear bottom
96-well plates, plate reader and for plasma and serum, Millipore
Microcon YM-10.
EXAMPLES
A rat serum sample was assayed in duplicate using the
standard 96-well protocol. The arginase activity was 322 ± 5 U/L. An
undiluted human serum from a healthy donor had an arginase activity of
0.88 ± 0.02 U/L.
PUBLICATIONS
[1]. Moertel, L. et al. (2008) Comparative real-time
PCR and enzyme analysis of selected gender-associated molecules in
Schistosoma japonicum. Parasitology 135, 575–583.
[2]. Pulichino, A.M. et al. (2008) Identification of
Transforming Growth Factor
β 1 – Driven Genetic Programs of Acute Lung
Fibrosis. Am. J. Respiratory Cell & Mol. Biol. 39: 324-336.
[3]. Ndolo, E.A. et al. (2010) The Role of Lysosomes
in Limiting Drug Toxicity in Mice. J Pharmacol Exp Ther. 333:120-128. |
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