Mouse GFAP ELISA Kit
Mouse GFAP ELISA Kit
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Product Specification
| Usage |
1. 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, 2. Sample processing and requirements: 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. If the sample tested is not among the sample types listed in the instructions, it is recommended to conduct pre-experiments to verify its detection effectiveness. Serum: Whole blood samples collected in serum separation tubes were placed at room temperature 2 Hour or 2-8℃ 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. Plasma: use EDTA Or heparin as an anticoagulant to collect a sample, and collect the sample 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. Tissue homogenate: With pre-cooled PBS(0.01M,pH=7.4) The tissue is flushed to remove residual blood (lysed red blood cells in the homogenate can affect the test results), and the tissue is weighed and cut into pieces. Combining the shredded 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 grind thoroughly on ice or grind in a homogenizer. 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. 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. Cell lysate: Precooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200μLPBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, take the supernatant for detection. Other biological samples: 1000×g Centrifugation 20 Minutes, take the supernatant to detect. Sample Appearance: The sample should be clear and transparent, and the suspended solids should be centrifuged to remove. Sample Preservation: 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 sample will affect the final test result, so hemolyzed samples are not suitable for this test. 3. Sample dilution plan: Please estimate the concentration range of the sample in advance. If your test sample needs to be diluted, refer to the dilution plan as follows: Dilution 100 Times: One step dilution. Take 5μL Sample to 495μL Within universal diluent, do 100 Double dilution; Dilution 1000 Times: Two-step dilution. Take 5μL Sample to 95μL Within universal diluent, do 20 Dilute, and then take 5μL20 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 1000 Times; Dilution 100000 Times: Three-step dilution. Take 5μL Sample to 195μL Within universal diluent, do 40 Dilute, and then take 5μL40 Double dilute sample to 245μL Within universal diluent, do 50 Time dilution, and finally take 5μL 2000 Double dilute sample to 245μL Within universal diluent, do 50 Double dilution, total dilution 100000 Times; The amount of liquid taken during each dilution step is not less than 3μL , the dilution factor is not more than 100 Times. Each step of dilution should be mixed evenly to avoid foaming. 4. 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 : join 1mL Universal Diluent into Lyophilized Standard , let stand 15 Minutes until it is completely dissolved and then gently mix ( The concentration is 8000pg/mL) And then according to the following concentrations: 8000pg/mL 、 4000pg/mL 、 2000pg/mL 、 1000pg/mL 、 500pg/mL 、 250pg/mL 、 125pg/mL 、 0pg/mL The dilution was performed. Double dilution method: Take 7 branch EP Tubes, each tube is added 500μL Universal diluent ,8000pg/mL Pipette from the standard working solution 500μL To the first EP Mix evenly in a tube 4000pg/mL According to this step, suck and mix the standard working solution in turn. The last tube is directly used as a blank hole, so there is no need to suck liquid from the penultimate tube, as shown in the figure below.
3 Preparation of biotinylated antibody detection working solution: before use 15 Minutes will 100× Concentrated biotinylation detection 1000×g Centrifugation 1 Minutes, with a universal diluent 100× Concentrated biotinylated detection antibody diluted into 1× Working concentration ( Example: 10μL Concentrate +990μL Universal diluent ) , now available for use. 4 Preparation of enzyme conjugate working solution: before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g Centrifugation 1 Minutes, with a universal diluent 100× The concentrated enzyme conjugate is diluted into 1× Working concentration ( Example: 10μL Concentrate +990μL Universal diluent ) , now available for use. 5 、 1× Wash liquid preparation: Take 10mL20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out from 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 ) 。 5. 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 100μL Each well is added to the corresponding well, and the blank well is added 100μL Universal diluent. After covering the sealing film 37℃ Incubation 60 Minutes. (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 Add biotinylated antibody detection: take out the enzyme labeled plate, discard the liquid, and do not wash it. Add directly to each well 100μL Biotinylated antibiotic detection working solution, after covering with sealing film 37℃ Incubation 60 Minutes. 4 Plate washing: discard the liquid and add to each well 300μL1x 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). 5 Add enzyme conjugate working solution: add each well 100μL Enzyme conjugate working solution, after covering with sealing membrane 37℃ Incubation 30 Minutes. 6 Plate washing: discard the liquid according to the steps 4 Washing method, wash plate 5 Times. 7 Add substrate: add per well 90μL Substrate (TMB) Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 8 Add stop solution: take out the enzyme label plate and add it directly to each well 50μL Stop solution, immediately in 450nm Wavelength measurement of each well OD Value. VI. 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. 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.
7. Kit performance: 1 Repeatability: the coefficient of variation in the plate is less than 10% , the interplate coefficient of variation is less than 10% 。 2 Recovery rate: added to selected healthy serum, plasma and cell culture supernatant 3 Mice at different concentration levels GFAP , calculate the recovery.
3 , linear dilution: respectively in the selected 4 High concentration mice were added to healthy serum, plasma and cell culture supernatant GFAP , linearity was assessed by dilution within the standard curve kinetic range.
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| Sensitivity | 63.12pg/mL | |||||||||||||||||||||||||||||||||||
| Theory | This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, HRP enzyme conjugate were sequentially added to microwells pre-coated with mouse glial fibrillary acidic protein (GFAP) capture antibody, incubated and washed in the middle, and colored with substrate TMB. TMB was converted to blue under the catalysis of peroxidase (HRP), and to final yellow under the action of acid. There was a positive correlation between the depth of color and the mouse glial fibrillary acidic protein (GFAP) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||||
| Synonym | Mouse Glial Fibrillary Acidic Protein (GFAP) ELISA Kit | |||||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||||
| Composition |
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| Background | Glial fibrillary acidic protein (GFAP) is a protein encoded by the GFAP gene. It is a type III intermediate filament (IF) protein expressed by many cell types of the central nervous system (CNS), including astrocytes and developing epithelial cells. It is closely related to three other non-epithelial type III IF family members-vimentin, norprotein and peripheral proteins, all of which are involved in the structure and function of the cytoskeleton. It is thought to help maintain the mechanical strength of astrocytes as well as the shape of the cells. The protein was named by Lawrence F. Eng in 1969, and it was first isolated and characterized. | |||||||||||||||||||||||||||||||||||
| Storage Temp. | Unopened kit, stored at 4 °C, shelf life 6 months. | |||||||||||||||||||||||||||||||||||
| Test Range | 125-8000pg/mL |
