IM-CMG
CMG035-140718
718E
FOR RESEARCHRESEARC USE ONLY
Catalog #CMG-0#035

MC Reagent Biochemical Assay Series

Magnesium Assay Kit
Xylidylblue
Xylidylblue-I Chromogenic method
Biochemical
Bioc
Significance and Test Summary
AlI ATP-dependent
ATP dependent enzyme require Mg2+ as a cofactor for the enzymes to react and maintain the electricalexcitability of the muscular and nervous cells. Regulation takes place mainly via the kidneys, especially vvia
ia theascending loop of Henle. A low magnesium level is found in malabsortion syndrome, diuretic or aminoglucosidetherapy.
therapy. Hypermagnesemia is found in acute and chronic renal failure, glomerulonephritis, Addisons’s disease orintensive anti acid therapy, magnesium excess, and magnesium release from the intracellular space.
This product is a direct colorimetric assay kit without deproteinization
eproteinization of the sample. Magnesium with Xylidylblue-I
Xylidylblue(as chelator) at alkaline pH, yields a purple colored complex. The intensity of the color forme
formedd is proportional tothe magnesium concentration in the sample.
sample1. Kit contents (100 tests)
R-1
RSTD

Chelate color
2 mg/dL
g/dL Mg Standard

1 x 25 mL
1 x 0.5 mL

Readyeady to use
Ready to use*Storage conditions: Store at 2-8°C.
2
Don’t freeze
freeze.
*Expiration:*Expiration: 1 year. After the vials are opened,opened, the kit should be used within one monthmonth.
*Measuring range: 0.05-3.5
3.5 mg/dL

2. Materials required but not provided(1) Distilled water(2) Micropipettors and pipette tips
(3) Clear flatflat-bottom 96-wellwell plate(4) Microplate reader with 660 nm capability
apability

3. Assay preparation
Bring allll reagents to room temperature before use.

4. Sample preparation
Serum/Serum/ Plasma: Insoluble substances in serum and plasma samples should be removed by filtration orcentrifugation. EDTA
EDTA-plasma
plasma cannot be used.
Urine (24 hour pooled urine)/ Dilute the sample 1/2 in distilled water. Add 6M HCl to the diluted sample andadjust pH 2.0
2.0-3.0 (e.g. 5--10 µL of 6M HCl/1
HCl/ mL of lysate). Centrifuge at 6,000 rpm for 15 min
minutes.. Collect thesupernatant and use it for assay.
Biological fluid: Add 6M HCl to the sample and adjust pH 2.0
2.0-3.0 (e.g. 5--10 µL of 6M HCl/HCl/1 mL of lysate).
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. Centr
Centrifuge
ifuge 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 5 µL of Distilled
istilled water(Blank)/STD(Standard)/
water(Blank)/STD(Standard)/Sample
ample into each well.
(2) Add 250 µµL of R-1
1 to each well and incubateinc
for 5 minutes
utes at room temperature.
temperature(3) Read the absorbance at 660 nm
nm. ----- OD

6.. Calculations
Calculations
ΔODStandard = ODStandard - ODBlank, ∆ODSample = ODSample - ODBlank
Magnesium (mg/dL) ==ΔODSample/ΔODStandard x 2
Magnesium (mM) =Δ
ΔODSample/Δ
ΔODStandard x 0.824(Assay example)
DW (Blank)
Standard
SampleOD (660 nm)
0.387
0.241
0.271

ΔOD
-0.146-0.116

Magnesium (mg/dL)(mg/1.59

ΔODStandard = 0.241 - 0.387 = -0.146
0.146
ΔODSample = 0.271 - 0.387 = -0.116
0.116
MagnesiumSample = ∆OD∆∆ODStandard x 2 = (-0.116/ -0.1460.146) x 2 = 1.59 (mg/dL)
Sample/∆OD
MagnesiumSample (mmM) = ∆ODSample/∆ODStandard x 0.824 = (-0.116/ -0.1460.146) x 0.824 = 0.655 (mM)

7.. Interferences
EDTA inhibits magnesium to chromogenic system. The test is not affected by presence of bilirubin-F
F andbilirubin-C
bilirubin up to 40 mg/dL, hemoglobin up to 1 g/dL and chyle up to 3000 FTU.
FTU

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

9.. Reference.
(1)1) Mann C. K, Yoe J. H : Spectrophotometric determination of magnesium with sodium
1-azo-2-haydroxy
haydroxy-3-(2,4(2,4-dimethyl-carboxanilicarboxanili-do)do)-naphtalene
naphtalene-1-(2-hydroxyhydroxy-benzenebenzene-5-sulphonate),sulphonate), AnalChem,, 28, p202p202-205
205 (1956)(22) Sakamoto. A, Terui. Y, Yamam
Yamamoto.
oto. T, Kasahara. T, Nakamura. M, Tomitori. H, Yamamoto. K, Ishihama A,Michael. AJ, Igarashi. K, Kashiwagi.
Kashiwagi. K : Enhanced biofilm formation and/or cell viability by polyamines throughstimulation of response regulators UvrY and CpxR in the two
two-component
component signal transducing systems, andribosome recycling factor
factor, Int J Biochem Cell Biol
Biol,, 44(15), p1877-1886
p1877 1886 (2012).
(201210.
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 densityensity. 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.
centrifugatio(6) 24 hour pooled urine
urine:: Dilute the sample 1/10 in distilled water. Mix. Multiply results by 10 (dilution factor) for assay sample.

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