Copper, Low Concentrate, Assay Kit

Product#: CU20ME
$520.00
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Copper, Low Concentrate, Assay Kit

Cat. No. CU20ME
Size 100 tests / 1 kit
Store oC
Measuring range  2 - 80 ug/dl
Wavelength (maximume wavelength) 570 - 590 nm (580nm)
Chromogen 3,5-DiBr-PAESA
Target Copper (Cu2+ , Cu+)


Description
 
Metallo Assay Copper Low Concetrate Assay Kit LS (DiBr-PAESA Method) can measure Copper ion (Cu2+, Cu+) in the biological samples such as Urine saliva, and wide range of samples by microplate reader

Physiological function of protein holding copper as a cofactor is a regulation of in-vivo redox status. Many of copper enzymes react directly with oxygen. 95% of copper in plasma is bonded with alpha-2-globulin, ceruloplasmin and oxidase of ferroxidase activity. Deficiency of copper causes cardiopathy, osteoporosis, osteoarthritis, Menkes syndrome, and Wilson's disease. It is widely known that copper deficiency lowers the anti-oxidant function in vivo. On the contrary, excessive dosage or consumption of copper is poisonous to the health. 

This product is a direct colorimetric assay kit without deproteinization of the sample. Dissociated copper from the copper binding protein or organic ligand by weakly acid buffer and reduced by means of reducing ascorbic acid (:Cu2+→Cu+). Cu+ ions give a blue colored complex with 3, 5-DiBr-PAESA (as chromogen). The color intensity is proportional to the amount of copper present in the sample.

Metallo Assay Copper Low Concentratekit LS is intended for the quantitative determination of copper in urine or saliva by 96-well reader use.

Notes
  1. Unstableness of incubation temperature may result in unstable results.
  2. Use disposable test tube and glassware washed with 1M HNO3 or 1M HCl solution and distilled water.
  3. Accuracy in pipetting volume for samples and reagents may affect the quality of assay. Please note that samples, standards and Working Reagent must be poured accurately µL level.
  4. Temperature for chromogen reaction may affect optical density. Please try to extend or shorten chromogen reaction time depending on room temperature.
  5. In the cell lysate or the tissue extract use as specimen, high concentration of proteins or lipid, may affect observed value. Please remove its by ultrafiltration or centrifugation.
  6. Heme-containing copper cannot be measured in this assay kit.

Expiration date and preservation conditions
Storage conditions: Store at 2-8°C. Don’t freeze.
Expiration: 1 year from the date of manufacture.
After the bottles are opened, the kit should be used in 1 month.

Features
  • Using microplate reader for assay, no need to use Atomic Absorption Spectrometry or ICP-OES Optical Emission Spectrometry, which have been mainly used so far.
  • Good correlation with the result of Atomic Absorption Spectrometry and ICP-OES.
  • High throughput measurement by using microplate reader.
  • Samples from any animal species can be measured.
  • Assay time : 10 minutes
  • 1 point calibration with blank – do not need multiple point measurement to make a standard curve (r^2=0.9999).
  • Kit does not contain any hazardous components like cyanide or azide as preservative.
  • Samples : Serum, Plasma, Urine*, Saliva, Cell lysate, Tissue extract, Hair extract, Water, etc.
Memo
The main role of proteins which contain Copper (Cu) is oxidation-reduction function. Most enzymes which contain copper directly react with free-oxygen. 95% of copper in plasma binds to oxidative enzyme, that has alpha-2 globulin, ceruloplasmin and ferroxidase activity.

It is reported that heart-related disease, brittle-bone disease, osteoarthritis, Menkes syndrome, and Wilson's Disease are caused by deficiency of copper. On the other hand, excess copper concentration is also toxic.
 
Assay Principle
This kit measures copper concentration by color change caused by chelate complex formulation of 3-5 DiBr-PAESA and copper. Copper combined to protein ceruloplasmin is denatured by weak acid and denaturant and then formulates Copper- 3-5 DiBr-PAESA chelate complex. Measuring this complex by 580 nm wavelength and obtain copper concentration.
CU03ME_fig1.jpg 

Sample Preparation
CU03ME_fig2.jpg 
  1. Samples which do not require preparation
    • e.g. Serum, Plasma
    • * EDTA treated sample is not suitable for this assay.
  2. Samples which require acid extraction
    • e.g. cell lysate, tissue homogenate and other liquid samples.
    • Measurable chemical species is limited as follows:
      • Free copper
      • Copper binds to protein (Protein-Cu2+, e.g. albumin, metallothionein)
    • Lysis buffer contains EDTA is not suitable for this assay.
    • Organo-copper and Heme-copper require acidolysis.
  3. Microwave method and samples which require acidolysis
    • e.g samples which contain strong chelate agents such as EDTA, Organo-copper (in the case of single bond -C-Cu-C-), copper in cyclic ligand (Cu-Porphyrin)
* After acidolysis, adjust sample pH in 2 to 3.

Handling Procedure

1. Apply DW, Standard and Sample (12 uL) to 96 well plate.


CU20ME_Fig1.jpg
 

2. Add working reagent (140 uL).


CU20ME_Fig2.jpg
 

3. Incubate at room temperature for 10 minutes


CU20ME_Fig3.jpg
 

4. Read the absorbance at 582 nm



 

5. Calculate concentration

ΔODStd = ODStd – ODBl, ΔODS = ODS – ODBl
Copper (μg/dL) = ΔODS/ΔODStd x 40

* ODS : ODsample, ODBl : ODblank, ODStd : ODstandard

 


Citation
  1. A novel method of preparing the monoform structure of catalytic antibody light chain  The FASEB Journal ; November 2, 2015
  2. Pheomelanin Formation and Low Tyrosinase Activity in Fading Body Color Variant BdlR Strain Oryzias latipes. J Life Sci ; June, 2014
  3. Cuprizone short-term exposure: astrocytic IL-6 activation and schizophrenia-like behavior. J Pharmacol Sci ; March 15, 2013

References
  1. Wilson S. A. K., Brain, 34, 295-309 (1912).
  2. Osman M. A., Patel R. B., Schuna A.,
  3. Sundstrom W. R., Welling P.G., Clin. Pharmacol. Ther., 33, 465-470 (1983).
  4. Taira K., Takagawa K., Okawa M., Yoshida H., Jpn. J. Pediatr. Med., 29, 139-144(1997).
  5. A. Abe et al, Clin. Chem., 35 (4), 552 (1989)  
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