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Human Renin ELISA Kit

Human Renin ELISA Kit

Catalog Number: abs551745 Brand: Absin
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$346 USD
$346 USD
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Product Details

Product Specification

Usage Own test equipment required for the experiment:
1 Plate reader ( 450nm ）
2 High-precision sampler and gun tip: 0.5-10uL 、 5-50uL 、 20-200uL 、 200-1000uL
3 、 37℃℃ thermostatic box
4 Distilled water or deionized water

Sample handling and requirements:
Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hours or 4℃ Stay overnight, then 1000×g centrifuge 20 Simply remove the supernatant for minutes, or place the supernatant in... -20℃ or -80℃ Store, but avoid repeated freezing and thawing.
Plasma: Use EDTA Alternatively, heparin can be used as an anticoagulant to collect specimens, and the specimens can be stored after collection. 30 Within minutes 2-8℃1000×g centrifuge 15 The supernatant can be taken for testing within minutes, or the supernatant can be placed in... -20℃ or -80℃℃ Store, but avoid repeated freezing and thawing.
Tissue homogenization: pre-cooled PBS （ 0.01M,pH=7.4 Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results), weigh and crush the tissue. Mix the broken tissue with the corresponding volume PBS (Generally based on 1 ： 9 The weight-to-volume ratio, such as 1g tissue sample correspondence 9mL of PBS The specific volume can be adjusted appropriately according to the needs of the experiment, and records should be kept. Recommended in PBS (Add protease inhibitor) to a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, the homogenized solution was placed in 5000×g centrifuge 5~10 Minutes, take the supernatant for testing.
Cell lysate: pre-cooled for adherent cells PBS Wash gently, then digest with trypsin, 1000×g centrifuge 5 Cells were collected after minutes; Suspension cells can be collected directly by centrifugation. Collected cells were pre-cooled PBS cleaning 3 Times, each 1×10^6 Add to individual cells 150-200uL PBS Resuspend (recommended in PBS Adding a protease inhibitor; If the content is very low, it can be reduced PBS Volume) and disrupt the cells by repeated freeze-thaw or ultrasound. The extract was placed in 2-8℃ ， 1500×g centrifuge 10 Minutes, take the supernatant for testing.
Cell culture supernatant: Please 1000×g centrifuge 20 The supernatant can be taken for testing within minutes, or the supernatant can be placed in... -20℃ or -80℃ Store, but avoid repeated freezing and thawing.
Other biological fluids: 1000xg centrifuge 20 Minutes, take the supernatant for detection.

Pre-test preparation:
1 Please advance 10 Remove the kit from the refrigerator and allow to equilibrate to room temperature.
2 Preparation of standard gradient working solution: Add 1mL Add the general diluent to the lyophilized standard and let stand. 15 After it has been completely dissolved for minutes, mix gently (concentration is 2000pg/mL ） , followed by the following concentrations: 2000pg/mL 、 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 31.25pg/mL 、 0pg/mL Dilute.
double ratio dilution method :take 7 branch EP tube Add to each tube 500uL Universal diluent ,2000pg/mL Absorb from the standard working solution 500uL To the first one EP Mix well in the tube 1000pg/mL standard working solution Following this step, the mixture is sucked and mixed sequentially. The last tube is used directly as a blank hole. There is no need to draw liquid from the penultimate tube, as shown in the figure below.

3 Preparation of biotinylated antibody working solution: Before use 15 The concentrated biotinylated antibody was placed in 1000×g   centrifuge 1 Minutes, with universal diluent  100× Concentrated biotinylated antibody diluted to 1× Working concentration (e.g.: 10uL   concentrate +990uL   Universal Diluent)    Ready to use.
4 Preparation of enzyme conjugate working solution: Before use 15 minutes will 100× Concentrated enzyme conjugate 1000×g   centrifuge 1 Minutes, with universal diluent 100× concentrate HRP   The enzyme conjugate is diluted into 1× Working concentration (e.g.: 10uL   concentrate +990uL   Universal Diluent)    Ready to use.
5 、 1× Washing liquid preparation: Take 10mL 20× washing liquid to 190mL  In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals are completely dissolved).

Operation steps:
1 From room temperature equilibrium 10 After a few minutes, remove the required strips from the aluminum foil bag, and seal the remaining strips back in a self-sealing bag. 4℃ 。
2 Sampling: Samples or standards of different concentrations were added according to 100ul Each well is added to the corresponding well, and the blank well is added. 100uL Universal diluent. After covering the sealing film 37℃ incubation 60 minutes. (Recommendation)   Minimally dilute the sample to be tested with a general-purpose diluent. 1 After doubling, it was added to the enzyme-labeled plate for testing. Thereby reducing the influence of matrix effect on the test results. Finally, when calculating the sample concentration, the corresponding dilution factor needs to be multiplied. Duplicate wells are recommended for all samples and standards to be tested.
3 Add biotinylated antibody: remove the enzyme label plate, discard the liquid, and do not need to wash. Add biotinylated antibody working solution directly to each well 100uL After covering with a sealing film 37℃ incubation 60 minutes.
4 Washing plate: Discard the liquid and add it to each well. 300uL 1x Washing liquid, let stand 1 After 10 minutes, remove the detergent, pat dry on absorbent paper, and repeat the washing process. 3 (You can also wash the board using a board washing machine).
5 Add enzyme conjugate working solution: Add enzyme conjugate working solution to each well. 100uL After covering with a sealing film 37℃ incubation 30 minutes.
6 Washing plate: Discard the liquid according to the steps. 4 Washing method, washing plate 5 Next.
7 Add substrate: Add substrate to each well ( TMB ） 90uL Cover with a sealing film, 37℃ Incubation in light 15 minutes.
8 Add stop solution: Remove the enzyme-labeled plate and add stop solution directly to each well. 50uL Immediately 450nm Measure each hole at the wavelength OD Value.

Calculation of experimental results:
Result Judgment:
1 Calculate the average of standard and sample replicates OD Value and subtract the blank hole OD Value as the correction value. With concentration as the horizontal axis, OD Value is ordinate Draw the standard curve of the four-parameter logic function on double-logarithmic coordinate paper.
2 If the sample OD Values above the upper limit of the standard curve should be diluted appropriately and retested and multiplied by the appropriate dilution factor when calculating the sample concentration.
Sensitivity 13.27pg/mL
Theory This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Into microwells previously coated with Renin capture antibodies, the sample, standard, biotin-labeled detection antibody, and HRPase conjugate were added in sequence, followed by incubation and washing, and developed with the substrate TMB, which was converted to blue under the catalysis of peroxidase (HRP) and finally to yellow under the action of acid. The depth of color was positively correlated with Renin in the sample. The absorbance (OD value) was measured using a microplate reader at a wavelength of 450 nm to calculate the sample concentration.
Synonym Human Renin ELISA Kit
Detection Type double antibody sandwich method
Composition
Name 9 6 T  match   set remark
pre-wrapped 96 well enzyme label plate 8 hole ×12 strip without
Standard 2 branch
Dilute according to directions
Universal diluent
2×20mL
without
Concentrated biotinylation antibody detection ( 100× ）  
120uL
Dilute according to directions
Concentrated enzyme conjugates ( 100× ）
120uL
Dilute according to directions
20× washing liquid
2×10mL
Dilute according to directions
bottom thing （ TMB ）
10mL
without
stopping fluid
6mL
without
sealing film
4 Zhang
without
instruction manual
1 portion
without
Background Renin, also known as angiotensinogenase, is secreted by cells adjacent to the glomeruli, which sense changes in renal perfusion pressure through stretch receptors on the blood vessel walls. Paraglomerular cells are also stimulated by signals from the macular region to release renin. The macula senses a change in sodium delivery to the distal tubules and responds to a decrease in tubular sodium load by stimulating renin release from cells adjacent to the glomerulus. The macular region and paraglomerular cells together form the paraglomerular complex. Renin secretion is also stimulated by the sympathetic nervous system, mainly through the activation of β1 adrenergic receptors.
General Notes 1. Incubate strictly according to the specified time and temperature to ensure accurate results. All reagents must reach room temperature of 20-25℃ before use. Refrigerate and store reagents immediately after use.
2. Incorrect washing of the board may lead to inaccurate results. Ensure that the fluid in the wells is sucked dry as much as possible before adding the substrate. Do not allow the microwells to dry out during incubation.
3. Eliminate residual liquid and fingerprints from the bottom of the plate, otherwise it will affect the OD value.
4. The substrate developing solution should be colorless or very light in color. Substrate solutions that have turned blue should not be used.
5. Avoid cross-contamination of reagents and specimens to prevent erroneous results.
6. Avoid direct exposure to strong light during storage and incubation.
7. No reactant should come into contact with the bleaching solvent or the strong gases emitted by the bleaching solvent. Any bleaching ingredient will destroy the biological activity of the reaction reagents in the kit.
8. Expired products must not be used, and ingredients with different product numbers and batch numbers must not be mixed.
9. Recombinant proteins from sources other than the 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 testing devices should be handled according to the prescribed procedures.
Storage Temp. Unopened kit, stored at 4°C with a shelf life of 6 months.
Test Range 31.25-2000pg/mL
Applications Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids

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Immunohistochemistry