Thiol Quantification Assay Kit

Catalog No. ABIN7384581

Product Details

Detection Method: Colorimetric

Detection Range: 0-16 μM

Application: Quantification (Q)

Purpose: The kit is designed to measure free thiols and disulfides in biological samples.

Sample Type: Cell Extracts, Plasma, Urine

Analytical Method: Quantitative

Specificity: Specific for the detection of thiols in a variety of different biological samples.

Characteristics: Thiol and Disulfide Assay Kit is recommended for estimations of oxidative stress levels in biological samples such as plasma. Since many proteins contain redox-sensitive free thiols1, the identification and quantification of their different re

Components: Plate and the reagents neccesary to perform the assay.

Material not included: Pipettes, reaction tubes, plate reader.

Application Details

Comment:

For native free thiols: 20μL, For disulfides: 40μL

Assay Time: 1 - 2 h

Plate: Uncoated

Reagent Preparation:

Reagents Preparation: Thiol and Disulfide Reagent B: Add exactly 4 mL of ultrapure water- methanol (50 % ) to the provided vial. This reagent is not stable: prepare daily and discard after use. The kit includes one vial for each 192 tests. Thiol and Disulfide Reagent D: Add exactly 4 mL of methanol to the provided vial. This reagent is not stable: prepare daily and discard after use. The kit includes one vial for each 192 tests.

Assay Procedure:

Thiols and Disulfide Standard: Add exactly 2 mL of diluent to the provided vial for a final concentration of 10 mM and dilute 1:10 for a final concentration of 1 mM. Then prepare different dilutions as shown below in Table 1 (see booklet). Performing the Assay: It is possible to calculate both the native (naturally reduced) and total (chemically reduced) free thiols in each sample. For the native free thiols: 1. Add 20 μL of sample/standard. 2.Add 20 μL of Thiol and Disulfide Reagent A and wait 10 min. 3. Add 220 μL of Thiol and Disulfide Reagent C and 20 μL of Thiol and Disulfide Reagent D. Wait 10 min. 4. Read the absorbance at 412 nm. For the total free thiols: 1. Add 20 μL of sample. 2.Add 20 μL of Thiol and Disulfide Reagent B and wait 10 min. 3. Add 220 μL of Thiol and Disulfide Reagent C and 20 μL of Thiol and Disulfide Reagent D. Wait 10 min. 4. Read the absorbance at 412 nm.

Calculation of Results:

1.Zeroed the absorbance values: ?A412 nm = A412 nm sample/standard - A412 nm blank. 2.Plot the zeroed absorbance (?A412 nm) of standards as a function of their final concentrations (Table 1). See Figure 2 for a typical standard curve. 3.Calculate the free thiols value of the samples using the equation obtained from the linear regression of the standard curve substituted ?A412 nm values for each sample. 4.The reduced thiols concentration in the sample is then the free thiols value calculation for the native free thiols assay. 5.The disulfides concentration in the sample results from the difference between the free thiols value calculations for the total free thiols assay and the native free thiols assay. Free Thiols (μM) = (?A412nm - intercept) / slope

Restrictions: For Research Use only

Handling

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C