Mouse GST ELISA Kit
Mouse GST ELISA Kit
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Product Specification
| Usage | Sample Processing and Requirements: Serum: Whole blood samples collected in serum separator tubes should be placed at room temperature for 2 hours or at 4°C overnight. Then, centrifuge at 1000×g for 20 minutes and remove the supernatant. Alternatively, the supernatant may be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Plasma: Specimens should be collected using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. The supernatant may be removed for testing, or the supernatant may be stored at -20°C or -80°C, but repeated freezing and thawing should be avoided. Tissue Homogenate: Rinse tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate may affect the measurement results). Weigh and mince the tissue. Mix the minced tissue with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse the tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. Cell Lysis Buffer: Adherent cells are gently washed with ice-cold PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells are washed three times with ice-cold PBS and resuspended in 150-200µL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freeze-thaw cycles or sonication. Centrifuge the extract at 1500 × g for 10 minutes at 2-8°C, and remove the supernatant for testing. Cell culture supernatant: Centrifuge at 1000 × g for 20 minutes, and remove the supernatant for testing. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000 × g for 20 minutes, and remove the supernatant for testing. Pre-test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 10 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is directly used as a blank well, and there is no need to aspirate liquid from the penultimate tube. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use, and dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent (e.g. 10uL concentrate + 990uL universal diluent), and use it immediately. 4. Preparation of Enzyme Conjugate Working Solution: 15 minutes before use, centrifuge 100 μL of concentrated enzyme conjugate at 1000 × g for 1 minute. Dilute the 100 μL concentrated HRP conjugate with universal diluent to a 1 μL working concentration (e.g., 10 μL concentrate + 990 μL universal diluent). Prepare freshly for use. 5. Preparation of 1 μL Wash Solution: Dissolve 10 mL of 20 μL Wash Solution in 190 mL of distilled water. (Concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing.) Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return them to 4°C. 2. Sample Addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the plate. This will minimize matrix effects on the test results. The sample concentration should be multiplied by the dilution factor when calculating the final sample concentration. It is recommended to run replicates for all samples and standards.) 3. Biotinylated Antibody Addition: Remove the plate and discard the liquid. Do not wash. Add 100 μL of biotinylated antibody working solution directly to each well. Cover with a film sealer and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x wash buffer to each well. Let stand for 1 minute. Shake off the wash buffer and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of enzyme conjugate working solution to each well, cover with a film sealer, and incubate at 37°C for 30 minutes. 6. Wash: Discard the solution and wash the plate five times according to the washing method in step 4. 7. Add Substrate: Add 90 μL of substrate (TMB) to each well, cover with a film sealer, and incubate at 37°C in the dark for 15 minutes. 8. Add Stop Solution: Remove the plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at 450 nm. Calculation of Experimental Results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction value. Plot a standard curve for the four-parameter logistic function on double-logarithmic graph paper with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a glutathione transferase (GST) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of glutathione transferase (GST) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||
| Synonym | Mouse Glutathione transferase ELISA Kit | |||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||
| Composition |
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| Background | Glutathione transferases (GSTs), formerly known as ligands, are a family of isoenzymes of eukaryotic and prokaryotic phase II metabolism, best known for their ability to catalyze the conjugation of the reduced form of glutathione (GSH) to xenobiotic substrates for detoxification. The GST family comprises three superfamilies: cell membrane, mitochondrial, and microsomal proteins—also known as MAPEGs. GSTs can comprise up to 10% of cell membrane proteins in some mammalian organs. GSTs catalyze the conjugation of GSH to electrophilic centers on a variety of substrates via sulfhydryl groups, rendering the compounds more water-soluble. GSTs can also bind toxins and function as transport proteins, leading to the early term for GSTs, ligandin. | |||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may lead to inaccurate results. Ensure that the wells are completely aspirated before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components from different product numbers and batches. 9. Recombinant proteins from sources other than the test kit may not match the antibodies in this kit and may not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and the samples and detection devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Store at 2-8°C. Valid for 6 months. | |||||||||||||||||||||||||||||||
| Test Range | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
