IM-CZN
CZN001-140718
718E
FOR RESEARCH USE ONLY
Catalog #CZN-001#001

MC Reagent Biochemical Assay Series

Zinc Assay Kit
5 Br-PAPS
PAPS Chromogenic method
Biochemical
Bioc mical Significance and Test Summary
Zinc is a cofactor of more than 200 kinds of metalloenzymes, and is also a trace element concerned in syntheticof ribonucleic acid or protein. It is widely known that acute zzinc
inc deficiency during the growth stage of themammalian, results in a severe impairment of the skin or hair, and may lead to arrested development. Zinc isessential to reproduction of cell and its relevant supply is necessary for healthy growth.
This product is a direct colorimetric assay kit without deproteinization
eproteinization of the sample. At alkaline pH,pH, in a buffered
media, zinc reacts with the specific complex, forms a stable colored complex. The color intensity is proportional tothe amount of zinc present in the ssample.
ample.

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

BufferChelate color
200 µg/dL
g/dL Zn Standard

1 x 23 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:*Expir: 1 year. After the bottles are opened,opened, the kit should be used within one monthmonth.
*Measuring range: 4.0-1,000000 µg/dL

2. Materials required but not provided(1) Distilled water(2) Micropipettors and pipette tips
(3) Clear flat--bottom 96-well
well plate(4) Microplate reader with 560 nm capability

3. Assay preparation(1) Bring all reagents to room temperature before use.
(2) Prepare working reagent: Mix 230 µL of R-1
1 and 5 µµL of R-2
R for 1 test.
(e.g.)) Preparation for 50 testssRR-1: 230 µL x 50 tests = 11.5 mL
RR-2: 5 µL x 50 tests
test = 250 µL
Mix 11.5 mL of R-1 and 250 µL of R-2
2 in a vessel.

4. Sample preparation
Serum/Plasma:Serum/Plasma: Insoluble substances in serum and plasma samples should be removed by filtration or
centrifugation. EDTA
EDTA-plasma
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 (e.g. 5-105µL
L of
6 M HCl/1 mL of lysatelysate).
). Centrifuge at 6,000 rpm for 15 min
minutesutes.. Collect the supernatant and use it for assay.
Tissue: Add 5% TCA solution, vovortex
tex for 1 minuteute and incubate for 30 minutes
minat 4-8°C.
8°C. Centrifuge at 6,000 rpm
for 15 minutes
minutes.. 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(Standardwater(Blank) TD(Standards)/Sample
amples into each well.
(2) Add 230 µµL of Working Reagent to each well and incubate for 5 minutes
utes at room temperature.
(3) Read the absorbance at 560 nm (550-580 nm)) and 700 nm (reference(reference wavelength)
wavelength). ----- OD

6.. Calculations
Calculations
ΔODStandard = ODStandard - ODBlank, ∆ODSample = ODSample - ODBlank
Zinc (µg/dL)
g/dL) = ΔODODSample/ΔOD
ODStandard x 200
Zinc (µM)M) = ΔODSample/ΔODStandard x 30.6
(Assay example
example)DW (Blank)
StandardSample

OD (560 nm)
0.066
0.196
0.113

OD (700 nm)
0.026
0.027
0.028

OD
0.040
0.169
0.085

ΔOD
OD
0.129
0.045

Zinc (µg/dL)
69.8(a) Measurement at 560 nm and 700 nm (reference wavelength):
OD = OD (560 nm) - OD (700 nm)
ΔODStandard = (0.196 - 0.027) - (0.066 - 0.026) = 0.129
ΔODSample = (0.113 - 0.028 ) - (0.066 - 0.026 ) = 0.045
ZincSample (µg/dL) = ΔODSample/Δ
ΔODStandard x 200 = (0.045/0.129
0.045/0.129) x 200 = 69.8 (µµg/dL)
ZincSample (µM) = ΔOD
ΔODStandard x 3
30.6 = (0.045/0.045/0.129) x 30.6 = 10.7 (µM)M)
Sample/ΔOD(b) Measurement at 560 nm:
ΔODStandard = 0.196 - 0.066 = 0.130
ΔODSample = 0.113 - 0.028 - 0.066 = 0.047
ZincSample (µg/dL) = ΔODSample/Δ
ΔODStandard x 200 = (0.047/0.1300.047/0.130) x 200 = 72.3 (µµg/dL)
ZincSample (µM) = ΔOD
ΔODStandard x 330.6 = (0.047/0.047/0.130) x 30.6 = 11.0 (µM)M)
Sample/ΔOD7.. Interferences
EDTA inhibits zinc to chromogenic system. The test is not affected by presence of Bilirubin
Bilirubin-F and Bilirubin-C
Bilirubin up to40 mg/dL and chyle up to 500
5 FTU.

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

9.. Reference
Makino.
Makino T, Saito
Saito. M, Horiguchi.
Horiguchi. D, and Kina. K : A highly sensitive calorimet
calorimetricric determination of serum zinc using
water-soluble
water soluble pyridylazo dyedye. Clinical Chimica Acta
Acta, 120, p127--135 (1982).

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 HNO 3 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 or centrifugation.
(6) Species of zinc
zinc-porphyrins
porphyrins cannot be analyzed using this assay kit.

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