Glutathione Assay Kit

Stock Code: 3586493
Manufacturer Part No: 354102-100T

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Descriptions

Analysis Note

Measurement of Total Mercaptans (RSH): Assay at 356 nmThe Calbiochem Glutathione Assay Kit can be used for the measurement of other mercaptans (RSH), which include GSH. The assay is carried out in the absence of reagent R2 at 356 nm. The absorbance at 356 nm is a linear function of [RSH] concentration in the sample, but it is not GSH-specific. If the sample essentially contains GSH and a single other mercaptan i.e., N-acetylcysteine, these two mercaptans can be assayed by using the same single sample for two measurements. The first measurement is made at 356 nm before the addition of reagent R2 as described below, and the second measurement is made, after the addition of R2, at 400 nm.Adjust the spectrophotometer absorbance to zero at 356 nm with buffer only (solution 3). The reaction mixture is prepared as in Section 3, with the omission of step 4. The absorbance (A) is measured at 356 nm.A standard curve must be prepared with the corresponding mercaptan in Table 2, to calculate the concentration. Figure 2 gives two examples of standard curves obtained with glutathione and Nacetylcysteine at 356 nm.GSH ConcentrationThe calculation is based on the following equation:[GSH] = {(A-A₀)/(E x l)} x Dwhere:[GSH] is the initial glutathione concentration in the sample, expressed as molar concentration.A and A₀ are the absorbances measured in the presence and in the absence of sample, respectively.E is the apparent molar extinction coefficient of the product measured at 400 nm.l is the optical path (cm).D is the dilution factor of the sample.Total Mercaptan ConcentrationThe calculation is based on the modified version of the equation used for GSH:[total RSH] = { (A - A₀) / (E x I) } x Dwhere :[total RSH] is the concentration of total mercaptan in the sample.A and A₀ are the absorbances measured in the presence and in the absence of sample, respectively.E is the apparent molar extinction coefficient of the product measured at 356 nm.I is the optical path (cm).D is the dilution factor of the sample.Note: Do not add reagents R1 and R2 in reverse order. The temperature, (25 ± 3°C), should be kept constant throughout the experiment. The final reaction volume (1 ml) should not vary from one measurement to another. The absorbance at 400 nm is proportional to glutathione concentration. It is stable for min, provided that the reaction mixture is kept in the dark. The sensitivity of the assay for mercaptans at 356 nm is of the same order of magnitude as that of glutathione at 400 nm as shown in Table 2.

Sensitivity: From 30 repeated measurements performed on the control ([RSH] = 0) on the same day, under the same experimental conditions, the detection limit of the assay was 5 µmol/l in the final reaction mixture (spectrophotometric cuvette). For example, using the maximum volume of 300 µl of a sample would therefore give a detection limit for glutathione of about 17 µmol/l.

Components

A Chromogen, 30% NaOH, Buffer, and a user protocol.

General description

Note: 1 T = 1 test.

Oxidative stress occurs in most, if not all, human diseases. Our understanding of the role played by reactive oxygen species in the progression of pathological changes is still evolving