GST alpha assay kit

GST alpha Assay Kit

Cat. Num. AAA480433

• Synonyms: GST alpha; GST alpha Assay Kit; Microplate Assay for GSTA; GST alpha assay kit

• Procedural Notes: 1. Reagents can be used immediately upon removal from refrigeration.; 2. To minimize errors in absorbance measurements due to handling, wipe the exterior bottom of the microplate wells with a lint-free paper towel prior to inserting into the plate reader.; 3. Do not save excess diluted reagents or standards for future use.
• Sample Collection and Preparation: Samples should be stored at -80 degree C and thawed just prior to use. Avoid repeated freeze/thaw cycles for best results. This assay was developed and validated with human serum samples, however it does crossreact with rat.
• Reagent Preparation: 1. Wash Buffer: Dilute the wash buffer 1:5 with dH2O. For example, if all the Wash Buffer will be used dilute the 40 mL with 160 mL of dH2O, giving a total final volume of 200 mL.; 2. Antibody-HRP: Immediately prior to use, dilute the needed amount 1:25 with Assay Buffer. For example, if all will be used, dilute the complete 440 muL with 10.66 mL of Assay Buffer, giving a total final volume of 11 mL.; 3. Standard: Perform a 1:100 dilution using Assay Buffer, giving a final concentration of 20 ng/mL. For example, add the 30 muL of Standard to 2.970 mL of Assay Buffer giving a total final volume of 3.0 mL.
• Sample Preparation: It is recommended to do multiple sample dilutions to ensure that the concentration falls within the accepted range for the assay. Samples are to be diluted in Assay Buffer. Dilution factors of 1:2 and 1:4 are normally sufficient.
• Standard Curve Preparation: Set up for the standard curve by labeling dilution tubes and dispensing the indicated volumes of Assay Standard; GST; Concentration; (ng/mL); Assay; Buffer; (L); Volume of; 20 ng/mL Standard; (L); Final Volume; (L); S6 20 - 1000 1000; S5 10 500 500 1000; S4 5.0 750 250 1000; S3 2.5 875 125 1000; S2 1.0 950 50 1000; S1 0.5 975 25 1000; B0 0.0 1000 - 1000 Buffer and diluted Standard according to Table 1 below.
• Preparation and Storage: 1. Store the components of this kit at the temperatures specified on the labels.; 2. Unopened reagents are stable until the indicated kit expiration date.

Related Product Information for GST alpha assay kit

Introduction: Glutathione S-Transferase (GST) has multiple isoforms. This assay is specific for Glutathione STransferase Alpha (GSTA) and is not known to cross react with the mu, pi, or theta variants. GSTA is a common biomarker for hepatocellular damage. 1 It also conjugates GSH to 4-hydroxynonenal, a product of lipid peroxidation2 and is an important player in cellular antioxidant defense mechanisms.3

Principle of assay: This is a standard sandwich enzyme-linked immunosorbent assay (ELISA). The plate is pre-coated with anti-GSTA and blocked, ready for the addition of samples and standards. The assay should take approximately 2.5 hours to run, plus any required sample preparation time. 1. Add 100 muL of Standards, Samples, or Blank (B0) to the corresponding wells on the microplate in duplicate. Incubate at room temperature for one hour.
2. Dump the contents of the plate and wash each well three times with 300 muL of Wash Buffer using a multichannel pipette. After the final wash, tap the plate on a lint-free paper towel to make sure there is no solution left in the wells.
3. Add 100 muL of the Antibody-HRP to each well. Incubate at room temperature for one hour.
4. Dump the contents of the plate and wash each well three times with 300 muL of Wash Buffer using a multichannel pipette. After the final wash, tap the plate on a lint-free paper towel to make sure there is no solution left in the wells.
5. Add 100 muL of TMB Substrate to each well. Allow color to develop for 30-40 minutes at room temperature.
6. Stop the reaction by adding 100 muL per well of 3N Sulfuric Acid (H2SO4).
7. Read the plate at 450 nm in a microplate reader

References

1. Vaubourdolle, M, et al., Clinical Chemistry 41: 1716-1719, 1995
2. Awasthi, Y. C, et al., Free Radic. Biol. Med. 37: 607-619, 2004
3. Yang, Y., et al. Toxicol. Appl. Pharmacol. 182: 105-115, 2002