Rat NGF ELISA Kit
Rat NGF ELISA Kit
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Product Specification
| protein | NGF | |||||||||||||||||||||||||||||||||||
| Usage |
Self-prepared test equipment required for the experiment: 1 , plate reader ( 450nm ） 2 , high-precision sampler and gun head: 0.5-10uL 、 5-50uL 、 20-200uL 、 200-1000uL 3 、 37℃℃ Incubator 4 Distilled water or deionized water, Sample handling and requirements: 1. The detection range of the kit is not equivalent to the concentration range of the test substance in the sample. It is recommended to estimate the concentration of the test substance in the sample through relevant literature before the experiment and determine the actual concentration of the sample through pre-experiment. If the concentration of the test substance in the sample is too high or too low, please dilute or concentrate the sample appropriately. 2. If the tested sample is not among the samples listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness. 3. Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 4℃ Overnight, then 1000×g Centrifugation 20 Minutes, take the supernatant, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. 4. Plasma: with EDTA Or heparin as an anticoagulant to collect specimens, and collect the specimens after collection 30 Within minutes 2-8℃1000×g Centrifugation 15 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. 5. Tissue homogenate: with 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 result), and break the tissue after weighing. Combine the fragmented tissue with the corresponding volume PBS (Generally according to 1 ： 9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and ground thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection. 6. Cell culture supernatant: please 1000×g Centrifugation 20 Minutes, take the supernatant to detect, or place the supernatant in -20℃ Or -80℃ Store, but repeated freezing and thawing should be avoided. 7. Other biological specimens: 1000xg Centrifugation 20 Minutes, take the supernatant to detect. 8. Sample appearance: The sample should be clear and transparent, and the suspended solids should be removed by centrifugation. 9. Sample storage: after sample collection, if 1 Those tested within weeks can be stored in 4℃ , if it cannot be detected in time, please pack it according to the one-time usage amount and freeze it in -20℃ （ 1 Within months), or -80℃ （ 6 Test within months) to avoid repeated freezing and thawing. Hemolysis of the specimen will affect the final test result, so hemolyzed specimens are not suitable for this test. Preparations before testing: 1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature. 2 , Standard gradient working solution preparation: add 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix (the concentration is 1000pg/mL ） And then according to the following concentrations: 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 62.5pg/mL 、 31.25pg/mL 、 15.625pg/mL 、 0pg/mL The dilution was performed. Double dilution method : Take 7 branch EP Tube , added to each tube 500uL Universal diluent ,1000pg/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 500pg/mL Standard Working Solution , according to this step, absorb and mix evenly in turn. The last tube is directly used as a blank hole , there is no need to suck liquid from the penultimate tube, as shown in the figure below.  3 、 HRP Preparation of anti-resistance detection working solution: before use 15 Minutes will concentrate HRP Antibody to 1000×g Centrifugation 1 Minutes, with a universal diluent 100× concentrate HRP The antibody is diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent), for same day use. 4 、 1× Wash liquid preparation: Take 10mL 20× Wash 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 left at room temperature and prepared after the crystals are completely dissolved). Operation steps: 1 Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。 2 , Adding samples: respectively add samples or different concentration standards 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 1 Hours. (Recommendation : Minimum dilution of sample to be tested with universal diluent 1 Times later, add the enzyme labeled plate for testing. So as to reduce the influence of matrix effect on the test results, and finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing). 3 Plate washing: discard the liquid and add to each well 300uL 1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (the plate can also be washed with a plate washing machine). 4 Add HRP Antibody: After washing the plate, add directly to each well HRP Antibody working solution 100μL , after covering the sealing film 37℃ Incubation 1 Hours. 5 Plate washing: discard the liquid according to the steps 3 Washing method, wash plate 5 Times. 6 Substrate addition: substrate is added per well ( TMB ） 90uL Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 7 Add stop solution: take out the enzyme label plate and directly add stop solution to each well 50uL , immediately in 450nm Wavelength measurement of each well OD Value. Calculation of experimental results: Result judgment: 1 , calculate the average of the standard and sample replica well OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD Value is ordinate , draw the standard curve of the four-parameter logic function on the double logarithmic coordinate paper (remove the values of the blank group when drawing). 2 If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration.  Kit Performance: 1. Repeatability: In-plate coefficient of variation is less than 10% , the interplate coefficient of variation is less than 10% 。 2. Recovery rate: added to selected healthy rat serum, plasma and cell culture supernatant 3 Rats at different concentration levels NGF , calculated recovery
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| Theory | This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with rat nerve growth factor (NGF) capture antibody, sample, standard product, and HRP-labeled detection antibody were sequentially added, incubated and washed in the middle, and colored by substrate TMB, which was converted to blue under the catalysis of peroxidase (HRP), and finally converted to yellow under the action of acid. There was a positive correlation between the depth of color and rat nerve growth factor (NGF) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. Sensitivity: 5.25 pg/mL Specificity: It can detect rat NGF in the sample without significant cross-reaction with its analogs. | |||||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||||
| Synonym | Rat Nerve Growth Factor ELISA Kit | |||||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Nerve growth factor (NGF) is a neurotrophic factor and neuropeptide, which is mainly involved in regulating the growth, maintenance, proliferation and survival of certain target neurons. Therefore, it is essential for the survival and maintenance of sympathetic and sensory neurons, because they undergo apoptosis in the absence of NGF. Since it was first isolated by Nobel laureates Rita Levi Montalcini and Stanley Cohen in 1956, many biological processes involving NGF have been identified, two of which are the survival of islet beta cells and the regulation of the immune system. NGF initially exists as a 7S, 130kDa complex when expressed, consisting of three proteins-Alpha-NGF, Beta-NGF and Gamma-NGF (ratio 2: 1: 2). This form of NGF is also known as proNGF (NGF precursor). The gamma subunit of the complex acts as a serine protease, cleaving the N-terminus of the beta subunit, thereby activating the protein into a functional NGF. The term nerve growth factor usually refers to the 2.5 S, 26kDa beta subunit of the protein, the only component of the 7SNGF complex that is biologically active (i.e. acts as a signaling molecule). | |||||||||||||||||||||||||||||||||||
| General Notes | 1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use. 2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation. 3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value. 4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized. 10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Unopened kit, stored at 4 °C, shelf life 6 months. | |||||||||||||||||||||||||||||||||||
| Test Range | 15.62-1000pg/mL | |||||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids |
