Thiol Detection Assay kit (100 tests)

Product#: TH01DE
$430.00
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Thiol Detection Assay

Cat. No. TH01DE
Size 1 Kit (100 tests)
Storage  4°C


Description

Thiol Detection Assay kit measures SH amount in sample by DTNB (Ellman) method.
10 minutes assay of Oxidative Stress by Elleman's MethodMetallogenics' Thiol Detection Assay Kit

In living organisms, thiol groups exist as free cysteine, glutathione, cysteine residues in proteins (such as albumin). Thiol groups attached to a carbon atom have a strong reducing ability, and plays an important role for the antioxidant capacity to scavenge ROS. According to a report, patients of various diseases exhibit low level of serum protein thiols. Furthermore, the oxidations of protein thiol groups contribute to the tertiary or quaternary structure of a protein. 

Metallogenics' thiol detection assay kit (Ellman's method) measures total thiol groups in blood samples. Ellman's reagent (DTNB: 5,5'-Dithiobis(2-nitrobenzoic acid)) reacts with thiol groups in blood samples, and yields TNB2- (5-Mercapto-2-nitrobenzoic acid), showing yellow color. The intensity of the color is proportional to the thiol groups in blood samples. Thiol concentration can be calculated by measuring absorbance of TNB2- at wavelength 412 nm (380 nm to 440 nm).
TH01DE_fig1.png 

The thiol detection assay kit (Ellman’s method) measures total thiol groups in the sample. Ellman’s reagent (DTNB: 5,5’-Dithiobis(2-nitrobenzoic acid)) reacts with thiol groups in the sample, and yields TNB2- (5-Mercapto-2-nitrobenzoic acid), that has yellow color. The intensity of the color formed is proportional to the thiol groups in the sample. Thiol concentration can be calculated by measuring absorbance of TNB2- at wavelength 412nm (380 nm to 440 nm).

Principle of Ellman's method

Ellman's reagent (DTNB:5,5'-Dithiobis (2-nitrobenzoic acid)) reacts with thiol group and produces 5-Mercapto-2-nitrobenzoic acid (TNB). Measure absorbance at λmax=412 nm and quantify thiol concentration.
TH01DE_fig2.png

Note

  • Use disposable test tube and glassware washed with 1 M HNO3 or 1 M HCl solution and distilled water.
  • 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.
  • 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.
  • Temperature for chromogen reaction may affect optical density. Please try to extend or shorten chromogen reaction time depending on room temperature.
  • Sample must be assayed fresh. If you stored the samples, please keep in -20 ℃.
  • Repeated freeze/thawing causes damage to sample.

Required but not provided

  • l Microplate reader (Wavelength 380-440 nm)
  • l Micro titer plate (96 wells)
  • l Pipette (20, 30, 200 µL)
  • l Pipette chips
  • l Distilled water

Operation

1. Sample preparation

◇Serum or Plasma
Insoluble solid in serum or plasma samples should be removed by filtration or centrifugation. EDTA-plasma is best suited.

2. Assay preparation

l R-A, R-B
Bring all reagents under room temperature before use.
l Reconstitute of STD Add distilled water to STD vial to dissolve N-acetyl-L-cysteine powder. (The volume of distilled water to add is indicated on the label of the vial.)

3. Assay procedure

Procedure using microplate reader.
(250 µL per 1 assay sample)
?Assay
(1) Add 20 µL of distilled water (Blank) / STD / sample into each well.
(2) Add 200 µL of R-A to each well.
(3) Read the absorbance at 412 nm. → OD1
(4) Add 30 µL of R-B to each well and incubate at room temperature for 10 min.
(5) Read the absorbance at 412 nm. → OD2

* Select the filter: 380-440 nm at 412 nm.

Expiration date and preservation conditions

Storage conditions: Store at 2-8°C. Don’t freeze.
Expiration: Use before 12 months from date of manufacture. After the bottles are opened, the kit should be used in 1 month.

Performance

Measuring range 0.05 – 2.0 mM
Imprecision Imprecision was evaluated using serum.

Within run

 

Mean mM

S.D

C.V %

Level 1

0.17

0.01

1.52

Level 2

0.26

0.01

3.47

 

Run to Run

 

Mean mM

S.D

C.V %

Level 1

0.21

0.00

1.31

Level 2

0.27

0.00

1.81

Feature

  • Rapid assay (Just 10 minutes)
  • Simple Protocol
  • No hazardous components such as cyanide or azide as preservative
  • Compatible with multi-species
  • Samples : Serum, Plasma
  • 2 points calibration

Reference

  1. Ellman GL “Tissue sulfhydryl groups.” Arch. Biochem. Biophys. 82 (1): 70–7. (1959).
  2. Hiroharu Einaga, Eiji Yoshihara, Yoshiyuki Matsuo, Junji Yodoi”Oxidative stress and redox regulation – protein oxidative modification and action-“ Journal of analytical Bio-Science, Vol.32, No4 (2009)
  3. Banne AF1, Amiri A, Pero RW. “Reduced Level of Serum Thiols in Patients with a Diagnosis of Active Disease. “Journal of Anti-Aging Medicine.Vol.6, Number 4, 2003 
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