Rat NGF ELISA Kit
Rat NGF ELISA Kit
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Product Specification
| Usage | Required Equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample Preparation and Requirements: 1. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. Before the experiment, it is recommended to estimate the analyte concentration in the sample based on relevant literature and conduct preliminary experiments to determine the actual concentration in the sample. If the analyte concentration in the sample is too high or too low, dilute or concentrate the sample appropriately. 2. If the sample being tested is not listed in the instructions, it is recommended to conduct a preliminary experiment to verify the validity of the test. 3. Serum: Incubate whole blood samples collected in serum separator tubes at room temperature for 2 hours or at 4°C overnight. Then, centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 4. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for analysis. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing. 5. Tissue Homogenate: Rinse the 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. Add the minced tissue to 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 tissue cells, the homogenate can be sonicated or repeatedly freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes and remove the supernatant for analysis. 6. Cell culture supernatant: Centrifuge at 1000×g for 20 minutes and remove the supernatant for analysis. Alternatively, store the supernatant at -20°C or -80°C, but avoid repeated freeze-thaw cycles. 7. Other biological specimens: Centrifuge at 1000×g for 20 minutes and remove the supernatant for analysis. 8. Sample appearance: The sample should be clear and transparent, and any suspended matter should be removed by centrifugation. 9. Sample Storage: Samples collected for testing within one week can be stored at 4°C. If testing cannot be performed promptly, aliquot the sample into single-use portions and store at -20°C (for testing within one month) or -80°C (for testing within six months). Avoid repeated freezing and thawing. Hemolysis of the sample can affect the final test results, so hemolyzed samples are not suitable for this test. Pre-Test Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and allow it to equilibrate to room temperature. 2. Prepare the gradient working solution of the standard: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 1000 pg/mL). Then dilute to the following concentrations: 1000 pg/mL, 500 pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, 31.25 pg/mL, 15.625 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 1000 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500 pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of HRP antibody working solution: Centrifuge the concentrated HRP antibody at 1000×g for 1 minute 15 minutes before use. Dilute 100× concentrated HRP antibody to a working concentration of 1× with universal diluent (e.g., 10 μL concentrate + 990 μL universal diluent) and use on the same day. 4. Preparation of 1× Wash Buffer: Dissolve 10 mL of 20× Wash Buffer in 190 mL of distilled water (Concentrated Wash Buffer removed from the refrigerator may crystallize, which is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing the buffer). 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 Loading: Add 100 μL of sample or standard of varying concentrations to the appropriate wells. Add 100 μL of Universal Diluent to the blank wells. Cover with a film sealer and incubate at 37°C for 1 hour. (Recommendation: Dilute the sample to be tested at least 1-fold with Universal Diluent before loading into the plate. This minimizes the impact of matrix effects on the test results. When calculating the sample concentration, multiply by the corresponding dilution factor. It is recommended to run replicates for all samples and standards.) 3. 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 microplate washer can also be used). 4. Add HRP Antibody: After washing, add 100 μL of HRP Antibody Working Solution directly to each well. Cover with a film sealer and incubate at 37°C for 1 hour. 5. Wash: Discard the liquid and wash the plate five times according to the procedure in step 3. 6. 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. 7. Add Stop Solution: Remove the ELISA 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: Interpretation of 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 the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis (excluding the blank well values when plotting). 2. If the sample OD value is above the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. 1. Repeatability: The intra-plate coefficient of variation is less than 10%, and the inter-plate coefficient of variation is less than 10%. 2. Recovery: Three different concentrations of rat NGF were added to the serum, plasma, and cell culture supernatant of healthy rats, and the recovery was calculated.
Theory |
This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Samples, standards, and HRP-labeled detection antibodies are added sequentially to microwells pre-coated with rat nerve growth factor (NGF) capture antibodies. After incubation and washing, the sample is developed with the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. The depth of the color is positively correlated with the rat nerve growth factor (NGF) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using a microplate reader to calculate the sample concentration. Sensitivity: 5.25pg/mL Specificity: It can detect rat NGF in the sample and has no obvious cross-reaction with its analogs. |
Source |
Rat |
Synonym |
Rat Nerve Growth Factor ELISA Kit |
Detection Type |
Double antibody sandwich method |
Composition |
Background |
Nerve growth factor (NGF) is a neurotrophic factor and neuropeptide that primarily regulates the growth, maintenance, proliferation, and survival of certain target neurons. Consequently, it is crucial for the survival and maintenance of sympathetic and sensory neurons, which undergo apoptosis in their absence. Since its initial isolation in 1956 by Nobel Prize winners Rita Levi-Montalcini and Stanley Cohen, numerous biological processes involving NGF have been identified, two of which are pancreatic beta-cell survival and immune system regulation. Upon expression, NGF is initially expressed as a 7S, 130 kDa complex composed of three proteins—Alpha-NGF, Beta-NGF, and Gamma-NGF (in a 2:1:2 ratio). This form of NGF is also known as proNGF (pro-NGF). The γ subunit of this complex acts as a serine protease, cleaving the N-terminus of the β subunit, thereby activating the protein into functional NGF. The term nerve growth factor usually refers to the 2.5S, 26 kDa beta subunit of the protein, which is the only biologically active (i.e., signaling molecule) component of the 7SNGF complex. |
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 result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly 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 agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. Storage Temp. |
If the unopened kit is stored at 4°C, the shelf life is 6 months. |
Test Range |
15.62-1000 pg/mL |
Applications |
Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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