Mouse TGF-beta 1 ELISA Kit
Mouse TGF-beta 1 ELISA Kit
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Product Specification
| Usage |
Need to bring your own test equipment 1. Microplate reader (can measure the absorption value of 450nm detection wavelength and the absorption value of 540nm or 570nm correction wavelength) 2. High-precision liquid dispenser and disposable tip 3. Distilled water or deionized water 4. Bottle washing (spray bottle), multi-channel plate washer or automatic plate washer 5. 500mL measuring cylinder 1. Preparation before the experiment 1. Sample collection and storage Cell culture supernatant: Particulate matter should be removed by centrifugation; Test the sample immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator at-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×). Serum: Samples were collected using a serum separation tube (SST) and the samples were placed at room temperature for 30 minutes. Centrifuge for 15 minutes at a rotation speed of 1000 g. The serum was removed immediately and tested immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator ≤-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×). Plasma: Plasma was collected using EDTA, heparin or citric acid as anticoagulant, centrifuged for 15 minutes within 30 minutes after collection at a rotation speed of 1000 g, and tested immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the one-time usage amount and store it in a refrigerator ≤-20 ℃ to avoid repeated freezing and thawing. Samples may need to be diluted with diluent (1 ×). 2. Preparation work before testing (Please place all reagents and samples at room temperature before use and let them stand15Minutes. All experimental samples and standards are recommendedDo repeat hole detection） 1 × Wash solution preparation:The concentrated washing liquid in the kit is 20 × mother liquid, which needs to be diluted into 1 × working liquid with distilled water before use.Example:Take 10mL of concentrated washing solution + 240mL of distilled water and make the volume to 200mL. In actual operation, the amount used can be calculated first, and then prepared. Color developer:According to 100 µ L per well, calculate the amount required for the current test, take out the corresponding volume of chromogenic agent, and protect it from light; The developer removed is for the same day use only. Buffer for dilution (1 ×):The kit contains buffer solution for concentration and dilution. Take 49.3 ml of 1X washing solution and add 0.7 ml of concentration and dilution buffer solution, and mix well to obtain 1X working solution. SA HRP:SA-HRP is 40 × mother solution. Before use, it needs to be diluted with dilution buffer (1 ×) to formulate 1 × working solution. The required amount per well is 100 µ L. Example: After 10 wells are used, 25 μL of 40 × mother liquor + 975 uL of dilution buffer (1 ×) is diluted to 1 mL to obtain 1 ml of 1 × working concentration detection antibody. Antibody detected:Refer to the bottle label for the redissolved volume of the test antibody and resuspend to the corresponding volume with the dilution buffer. Standard:Refer to the bottle label for the redissolved volume of the lyophilized standard to obtain a concentration of 2,000 pg/mL standard mother solution. Gently shake for at least 15 minutes and it dissolves well. (The standard has been activated and no reactivation is required) Add 500 μL of diluent (1 ×) to each dilution tube. Make serial dilutions of the standard mother liquor according to the figure below, and each tube must be thoroughly mixed before pipetting to the next tube. The mother standard solution without dilution can be used as the highest point of the standard curve (2,000 pg/mL) and the dilution (1 ×) can be used as the zero point of the standard curve (0 pg/mL).  Activation before sample loading
PS: Fetal calf and calf serum contain high TGF-β1, which can be as high as about 16 ng/ml. If the cell culture supernatant contains 10% fetal bovine serum, there will be a background TGF-β1 expression with a maximum content of about 1600 pg/ml. The blank medium control group can be added as a background subtraction, or serum-free medium can be directly selected to culture cells. 2. Experimental operation steps2. Take out the microplate from the sealed bag that has been balanced to room temperature. Please put the unused slats back into the aluminum foil bag and reseal them; 3. Add different concentrations of standard materials, experimental samples or quality control materials to the corresponding wells, 100 mL per well. The reaction wells were sealed with plate sealing tape and incubated at room temperature for 2 hours. 4. Suck off the liquid in the plate and use a bottle washer, a multi-channel plate washer or an automatic plate washer to wash the plate. 300 mL of washing solution was added to each well and the washing solution in the plate was aspirated off. Repeat the operation 3 times. Trying to absorb the residual liquid as much as possible every time you wash the plate will help to get good experimental results. At the end of the last plate washing, please suck all the liquid in the plate or turn the plate upside down, and pat all the residual liquid dry on absorbent paper; 5. Add 100 mL of detection antibody to each well. Seal the reaction wells with plate sealing tape, and incubate at room temperature for 2 hours; 6. Repeat the plate washing operation in step 4; 7. Add 100μL of diluted streptavidin-HRP to each microwell and incubate at room temperature for 20 minutes. Be careful to avoid light; 8. Repeat the plate washing operation in step 4; 9. Add 100 mL of chromogenic substrate to each microwell and incubate at room temperature for 5-30 minutes (under normal circumstances, the incubation time is about 15 minutes will get better results, but please carefully choose the time to terminate color development according to the actual situation of the experiment). Be careful to avoid light; 10. Add 50 mL of stop solution to each well, and the color of the solution in the well will change from blue to yellow. If the color of the solution changes to green or the color changes are inconsistent, pat the microplate gently to mix the solution evenly; 11. Within 30 minutes after adding the stop solution, use a microplate reader to measure the absorbance value of 450 nm, and set 540 nm or 570 nm as the correction wavelength. If dual-wavelength correction is not used, the accuracy of the results may be affected; 12. Calculation results: Average the corrected absorbance values (OD450-OD540/OD570) and multiple well readings of each standard and sample, and then subtract the average zero standard OD value. Four-parameter logic (4-PL) curve fitting was performed using computer software to create the standard curve. Alternatively, a curve can be generated by plotting the logarithm of the standard concentration versus the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process can generate a data fit that is sufficiently useful but less accurate. If the sample is diluted, the concentration should be calculated by multiplying the dilution factor.  1. Sensitivity: The lowest measurable value of TGF-beta 1 in mice is generally less than 6.8 pg/mL. The lowest measurable value is the corresponding concentration calculated from the mean of the zero-point absorbance values of 20 standard curves plus two standard deviations. 2. Calibration: This ELISA kit was calibrated via high purity recombinant mouse TGF-beta 1 protein expressed by CHO cells. 3. Linearity: Six different samples were spiked with high concentrations of mouse TGF-beta 1, and then the samples were diluted to the detection range with diluent (1 ×) to determine their linearity. 4. Analysis of frequently asked questions 1. Whiteboard (after the color development is completed, no color appears) < td style = "width: 45.9864%; text-align: center; height: 22 px; "> Solution
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| Synonym | CEDLAP, DPD1, latency-associated peptide, TGF beta1, TGFB, TGFB1, TGF-beta 1 protein, TGFbeta 1, TGF-beta 1, TGFbeta, TGF-beta-1, transforming growth factor beta-1, transforming growth factor, beta | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Description |
1. Detection principleDouble antibody sandwich ELISA was used in this experiment. Anti-mouse TGF-beta1 monoclonal antibody is coated on a microplate, and TGF-beta1 in samples and standards will bind to the antibody immobilized on the plate, and the free components will be washed away; Biotin-labeled anti-mouse TGF-beta 1 polyclonal antibody was added to bind to TGF-beta 1 bound to the microplate, and the free components were washed away; Streptavidin-labeled horseradish peroxidase that specifically recognizes biotin is added to form a complex. After washing away the free components, the substrate solution (color developer) is added, the color of the solution gradually turns blue, and the stop solution is added to the solution turns yellow and stops changing. The absorbance was measured with a microplate reader. 2. Product composition
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| Background | Transforming growth factors (TGF) Beta 1, 2, and 3 (TGF-beta 1, TGF-beta 2, and TGF-beta 3) are highly pleiotropic cytokines that can be secreted by almost all cell types. TGF-beta molecules have been proposed as cellular switches that regulate processes such as immune function, proliferation, and epithelial-mesenchymal transition. The targeted deletion of these genes in mice suggests that each TGF-beta isoform has certain non-redundant functions: TGF-beta1 is involved in hematopoiesis and endothelial cell differentiation; TGF-beta2 affects the development of the heart, lung, craniofacial, limb, eye, ear, and genitourinary system; TGF-beta 3 affects palatal organ development and lung development. The full in vitro biological activity of TGF-beta 5 has not yet been fully explored. However, in inhibitory bioassays, TGF-beta 1, TGF-beta 2, TGF-beta 3, and TGF-beta 5 have been found to be largely interchangeable, and TGF-beta 5 is expected to show a similar range of activities among other TGF-beta family members. To date, the production of TGF-beta5 has been demonstrated only in Xenopus laevis. The TGF-beta ligand is initially synthesized as a precursor protein undergoing proteolytic cleavage. The mature segment forms an active ligand dimer through a disulfide-rich core consisting of a characteristic "cysteine knot". TGF-beta signaling begins with complex binding to the accessory receptor beta glycan (also known as TGF-beta RIII) and the type II serine/threonine kinase receptor (known as TGF-beta RII). This receptor then phosphorylates and activates the type I serine/threonine kinase receptor, namely ALK-1 or TGF-beta RI (also known as ALK-5). Activated type I receptors phosphorylate and activate transcription-regulating Smad proteins. Differential responses to TGF-beta can be observed in different settings using other signaling pathways that are independent of Smad. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| General Notes | 1. Only for scientific research, not for in vitro diagnosis; 2. Please use it within the validity period of the kit; 3. The components of different kits and kits with different batch numbers cannot be mixed; 4. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with diluent (1 ×) and re-tested; If the cell culture supernatant sample needs to be distributed and diluted, cell culture medium can be used for other intermediate dilutions except for the last step of dilution with diluent; 5. Differences in test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipette, the plate washing technique, the reaction time or temperature, the storage of the kit, etc. 6. The termination solution in the kit is acidic solution. Please protect your eyes, hands, face and clothes when using it. |
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| Test Range | 31.3pg/mL-2000pg/mL |
