Human glutathione S transferase P1 (GSTP1) kit (ELISA) activity assay method

l This kit allows for the quantitative in vitro detection in serum, plasma, tissue homogenates and other biological fluids of human activity of glutathione S-transferase P1 (GSTP1) is.
l Validity period: 6 months
l Storage conditions: 2 -8 ° C
l This kit is for scientific research purposes only and should not be used for clinical diagnosis.
Experimental principle <br> The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules of the human glutathione S transferase P1 (GSTP1) capture antibody were pre-coated, and the specimen, the standard, and the HRP-labeled detection antibody were sequentially added, and the cells were washed and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with human glutathione S transferase P1 (GSTP1) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.

Sample processing and requirements
1. Serum : The whole blood sample collected in the serum separation tube is allowed to stand at room temperature for 2 hours or 4 ° C overnight, then centrifuged at 1000 × g for 20 minutes, the supernatant is taken, or the supernatant is placed at -20 ° C or -80 Store at °C, but avoid freezing and thawing repeatedly.
2. Plasma : specimens were collected with EDTA or heparin as anticoagulant, and the specimens were centrifuged at 1000 × g for 2 minutes at 2-8 ° C for 30 minutes after collection, and the supernatant was taken for detection, or the supernatant was placed. Store at -20 ° C or -80 ° C, but avoid repeated freezing and thawing.
3. Tissue homogenization: Wash the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood (the red blood cells in the homogenate will affect the measurement results), and then weigh the tissue after weighing. The shredded tissue and the corresponding volume of PBS (generally in a weight ratio of 1:9, such as 1g of tissue sample corresponding to 9mL of PBS, the specific volume can be adjusted according to the needs of the experiment, and record. Recommended to add in PBS The protease inhibitor) was added to a glass homogenizer and thoroughly ground on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, the homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was taken for detection.
4. Cell culture supernatant or other biological specimens: centrifuge at 1000 × g for 20 minutes, take the supernatant to detect, or store the supernatant at -20 ° C or -80 ° C, but avoid repeated freezing and thawing.
Note: Specimen hemolysis will affect the final test results, so hemolysis specimens should not be tested.

Reagents and equipment that are not provided

• Microplate reader (450nm)
• High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL
• 37 ° C incubator
• Distilled or deionized water

Kit composition

name	96-well configuration	48-hole configuration	Remarks
Microporous plate	12 holes × 8	12 holes × 4	no
Standard	0.3mL*6 tube	0.3mL*6 tube	no
Sample diluent	6mL	3mL	no
Detection antibody-HRP	10mL	5mL	no
20× washing buffer	25mL	15mL	Dilute according to the instructions
Substrate A	6mL	3mL	no
Substrate B	6mL	3mL	no
Stop solution	6mL	3mL	no
Sealing film	2 sheets	2 sheets	no
Instruction manual	1 serving	1 serving	no
Ziplock bag	1	1	no

Remarks:
1. The concentration of standard products is: 1600, 800, 400, 200, 100, 50 mIU/L
2. After a large number of normal specimen tests, the normal concentration values ​​of the specimens are within the detection range provided by the kit, and 50 μL of the sample can be directly loaded during the experiment. When part of the sample value exceeds the maximum standard concentration, the sample may be diluted with the sample dilution before the experiment.

Precautions

• Incubation is carried out strictly in accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C before use. Store the reagents refrigerated immediately after use.
• Incorrect cleaning can result in inaccurate results. Make sure to drain the liquid in the well as much as possible before adding the substrate. Do not let the micropores dry during the incubation.
• Eliminate residual liquid and fingerprints at the bottom of the board, otherwise it will affect the OD value.
• The substrate coloring solution should be colorless or very light, and the substrate liquid that has turned blue cannot be used.
• Avoid cross-contamination of reagents and specimens to avoid erroneous results.
• Avoid direct exposure to strong light during storage and incubation.
• After balancing to room temperature, the sealed bag was opened to allow water-repellent drops to coagulate on the cold strip.
• Any reagents should not be exposed to strong gases emitted by bleaching solvents or bleaching solvents. Any bleaching component will destroy the biological activity of the reagents in the kit.
• Expired products cannot be used.
• If the disease is likely to spread, all samples should be managed and the sample and test device processed in accordance with the prescribed procedures.

Reagent Preparation <br> The kit should be taken out of the refrigerated environment and should be equilibrated at room temperature before use.
Dilution of 20× Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20× wash buffer plus 19 parts of distilled water.

Steps

1. The required slats were taken out from the aluminum foil pouch after equilibrating for 20 min at room temperature, and the remaining slats were sealed back to 4 ° C with a ziplock bag.
2. Set standard and sample wells, standard wells with different concentrations of standard 50μL;
3. Add 50 μL of the sample to be tested to the sample well; blank holes are not added.
4. In addition to the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing plate, and incubated at 37 ° C in a water bath or incubator for 60 min.
5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution (350 μL), let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine) .
6. 50 μL of each of the substrates A and B was added to each well, and incubated at 37 ° C for 15 min in the dark.
7. 50 μL of the stop solution was added to each well, and the OD value of each well was measured at a wavelength of 450 nm within 15 min.

Calculation of experimental results <br> The OD value of the measured standard is the abscissa, the concentration of the standard is the ordinate, the standard curve is drawn on the coordinate paper or with the relevant software, and the linear regression equation is obtained, and the OD value of the sample is obtained. Substituting the equation and calculating the concentration of the sample.

Kit performance

• Detection range: 50 mIU/L – 1600 mIU/L.
• Sensitivity: The minimum detection concentration is less than 10 mIU/L.
• Specificity: Does not cross-react with other soluble structural analogs.
• Repeatability: The intra-plate variation coefficient is less than 10%, and the inter-plate variation coefficient is less than 15%.

Explanation <br> Due to the existing conditions and scientific and technical level, it is not possible to comprehensively identify and analyze all the raw materials provided by all suppliers. This product may have certain quality and technical risks.

• The final experimental results are closely related to the effectiveness of the reagents, the experimenter's related operations, and the experimental environment at the time. Be sure to prepare sufficient specimen backups.
• Different batches of the same product may have a slight difference, such as: detection limit, sensitivity and color development time, etc., please carry out the experimental operation according to the instructions in the kit. The electronic version of the website is for reference only.
• Only the reagents used in this kit can be used to ensure the test results. Do not mix products from other manufacturers. The best test results will only be obtained if the experimental instructions of this kit are strictly followed.
• The company is only responsible for the kit itself, and is not responsible for the sample consumption caused by the use of the kit. Please fully consider the possible usage of the sample before use, and reserve sufficient samples.
• Tissue homogenates or cell extracts prepared using chemical lysates may cause bias in ELISA results due to the introduction of certain chemicals.
• If the sample is a cell culture supernatant, there are many factors that interfere with such samples, such as cell status, cell number, sampling time, etc., so there may be cases where detection is not possible.
• Certain natural or recombinant proteins, including prokaryotic and eukaryotic recombinant proteins, may not be detected because they do not match the detection and capture antibodies used in this product.

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