IM-CCU
CCU400-140718
718E
FOR RESEARCH USE ONLY
Catalog #CCU-400#400

MC Reagent Biochemical Assay Series
3,5-DiBr
3,5
PAESA Chromogenic method

Copper Assay Kit
Biochemical
Bioc mical Significance and Test Summary
Physiological function of protein holding copper as a cofactor is a regulation of in-vivoin vivo redox status. Many of copper enzymes
react directly with oxygen. 95% of copper in plasma is boboundnd with ceruloplasmin (ferroxidase
ferroxidase). Deficiency of copper causes
cardiopathy, osteoporosis, osteoarthritis, Menkes syndrome, and Wilson's disease. It is widely known that copper deficiency
lowers the anti
anti-oxidantoxidant function in vivo.. On the contrary, excessiv
excessivee dosage or consumption of copper is poisonous to thehealth.
This product is a direct colorimetric assay kit. Dissociated copper from the ceruloplasmin
eruloplasmin-copper
copper complex by weakly acidbuffer and reduced by means of reducing ascorbic acid (:Cu 2+→Cu+). Cu+ ions give a blue colored complex with
3,5-DiBr-PAESA
3,5PAESA (as chromogen). The color intensity is proportional to the amount of copper present in the sample.

1. Kit contents (100 tests)
test
R-1
R-2
STD

Buffer (with reductant)
Chelate color
200 µg/dL
g/dL Cu Standard

1 x 24 mL
1 x 0.5 mL
1 x 1.2 mL

Ready to use
Ready to use
Ready to use*Storage conditions: Store at 2
2-8°C. Don’t freeze.
freeze*Expiration: 1 year. After the vials are oopened,pened, the kit should be used within one month.
month*Measuring range
range: 3-400 µg
g/dL

2. Materials required but not provided(1) Distilled water(2) Micropipettors and pipette tips
(3) Clear flat-bottom
flat bottom 96
96-wellwell plate(4) Microplate reader with 55880
0 nm capability

3. Assay preparation(1) Bring all reagents to room temperature before use.
(2) Prepare enough working reagent: Mix 240
2 µL of R
R-1 and 5 µL of R-2 for 1-test
test.
(e.g.) Preparation for 50 tests
test
R-1
1: 230 µL x 50 tests
test = 12.0 mL
R-2:2: 5 µL x 50 tests
test = 250 µL
LMix 112.0 mL of R-1
R and 250 µL
L of R-2
2 in a vessel.

4. Sample preparation
Serum/Plasma: Insoluble substances in serum and plasma samples should be removed by filtration or centrifugation.
EDTA-plasma
EDTA plasma cannot be used.
Urine (24 hour pooled urine)/urine)/Biological
Biological fluid: Add 6M HCl to the sample and adjust pH 2.0-3.0
2.0 3.0 (e.g. 55-10µL
L of 6M HCl/1mL
1mLof lysate).
lysa ). Centrifuge at 6,000 rpm for 15 minutes.
min. Collect the supernatant and use it for assay.
Tissue: Add 5% TCA solution, vovortex for 1 minuteminand incubate for 30 min
minutes at 4-8°C.
4. Centrifuge at 6,000 rpm for 15minutes.
min. Collect the supernatant and use it for assay.
Note: Sample pH should be between pH 2 and pH 8.

5. Assay protocol(1) Add 12 µL of Distilled water (Blank)/STD (Standard)/(Standard)/Sample
ample into each well.
(2) Add 240 µL
L of Working Reagent to each well and incubate for 10 minutes
minat room temperature.
(3) Read the absorbance at 5580 nm (570--590 nm) and 700 nm ((reference
reference wavelength
wave ength). ----- OD

6. Calculations
ΔODStandard = ODStandard - ODBlank, ∆ODSample = OD Sample - ODBlank
Copper (µµg/dL) = ΔODODSample/ΔODStandard x 200
Copper (µµM)=ΔOD
ODSample/ΔODStandard x 31.4(Assay example)
DW (Blank)
Standard
SampleOD (580 nm)
0.067
0.155
0.097

OD (700 nm)
0.028
0.053
0.037

OD
0.039
0.102
0.060

ΔOD
OD
0.063
0.021

Copper (µg/dL)(66.7(a) Measurement at 580 nm and 700 nm (reference wavelength):
OD = OD (580 nm) – OD (700 nm)
ΔODStandard = (0.155 - 0.053) – (0.067 - 0.028) = 0.063
ΔODSample = (0.097 - 0.037) – (0.067 - 0.028) = 0.021
CopperSample (µg/dL) = ∆OD∆ Sample/∆OD∆ODStandard x 200 = (0.021/0.021/ 0.063)
0.063 x 200 = 66.7 (µg/dL)
CopperSample (µM) = ∆ODSample/∆ODStandard x 31.4 = (0.021/0.021/ 0.063)
0.063 x 31.4 = 10.5 (µM)(b) Measurement at 580 nm:
ΔODStandard = 0.155 - 0.067 = 0.088
ΔODSample = 0.097 - 0.067 = 0.030
CopperSample (µg/dL) = ∆OD∆ Sample/∆OD∆ODStandard x 200 = (0.030/0.030/ 0.088)
0.088 x 200 = 68.2 (µg/dL)(CopperSample (µM) = ∆ODSample/∆ODStandard x 31.4 = (0.030/0.030/ 0.088)
0.088 x 31.4 = 10.7 (µM)
(7.. Interferences
EDTA inhibits copper to chromogenic system. The test is not affected by presence of bilirubin-F and bilirubin-C
C up to 40 mg/dL.

8. Quality Control
Use of control sera is recommended to monitor the quality of assay results.

9.. Reference(1)1) Abe. A,, Saito. Yamashita. S, Noma. A: Sensitive, Direct Colonmetric Assay for Copper in Serum
Serum. Clinical Chem,Chem 35(4),p552-554
554 (1989(1989).
(2)2) Sakamoto. A
A, Terui. Y, Yamamoto. T,T Kasahara. T,T Nakamura. M
M, Tomitori. H,H Yamamoto. K,
K Ishihama. A
A, Michael. A. J,
J
Igarashi. K, Kashiwagi. K : Enhanced biofilm formation and/or cell viability by polyamines through stimulation of response
regulators UvrY and CpxR in th
the two-component
component signal transducing systems, and ribosome recycling factor, Int J Biochem
Cell Biol. 44(11), p1877
p1877-86(2012).
86(2012).

10.
10. Technical support & troubleshooting(1) Unstableness of incubation temperature may result in unstable results.
(2) Use disposable test tube and glassware washed with 1M HNO3 or 1M HCl, and rinse with distilled water.
(3) Accuracy to the microliter is important to obtain good results. Ensure maximum precision when pipetting.
(4) Temperature for the chromogenic
chromogen reaction may affect the optical density.
density It may be necessary to adjust the reaction timedepending on the room temperature.
(5) HighHigh concentration of proteins or lipid inn cell lysate or in tissue extract may affect the observed value. Please remove themby ultrafiltration
ltration or centrifugation.
(6) Species of copper
copper-porphyrins
porphyrins cannot be analyzed using this assay kit.

Japan Institute for the Control of Aging (JaICA),
Nikken SEIL CCo., Ltd.
Haruoka 710-1,710 1, Fukuroi, Shizuoka 437
437-0122,0122, Japan
TEL: +81-538-49+8149-0125 FAX:
FAX +81-538-49
49-1267
www.jaica.com/e/ E-mail: biotech@jaica.com(Supported languages: Japanese and English)