Bovine β-lactoglobulin (β-Lg) Quantitative Detection Kit (ELISA) Instruction Manual [Kit name] Bovine β-lactoglobulin (β-Lg) Quantitative Detection Kit (ELISA) [Kit use] Quantitative detection of bovine serum , The content of β-lactoglobulin (β-Lg) in plasma and related liquid samples. [Detection principle] This kit adopts double antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the test sample to the pre-coated bovine β-lactoglobulin (β-Lg) antibody transparent enzyme label coated plate. After incubation for a sufficient time, wash to remove unbound components, and then add the enzyme label working solution After sufficient incubation time, wash to remove unbound components. Substrates A and B are added in sequence, and the substrate (TMB) is converted into a blue product catalyzed by horseradish peroxidase (HRP), which turns yellow under the action of acid. The protein (β-Lg) concentration was positively correlated. The OD value was measured at a wavelength of 450 nm. Based on the OD value of the standard and the sample, the content of bovine β-lactoglobulin (β-Lg) in the sample was calculated. [Kit composition] 1 enzyme label coated plate 12 wells × 8 strips 7 Developer B solution 6mL 2 standard 0.3mL * 6 tubes 8 stop solution 6mL 3 20-fold concentrated washing solution 25mL 9 instructions 1 part 4 biotin 1mL 10 seals 2 sheets of membrane 5 enzyme labeling reagent 10mL 11 sealed bag 1 6 developer A solution 6mL Remarks: The concentration of the standard product (S5 → S0) is: 200, 100, 50, 25, 12.5, 0 μg / mL. [ Required reagents and equipment not provided】 1, 37 ℃ incubator 2, standard microplate reader 3, precision pipette and disposable pipette tip 4, distilled water 5, disposable test tube 6, absorbent paper [operation steps] 1 2. Preparation: Remove the kit from the refrigerator and re-equilibrate at room temperature for 30 minutes. 2. Mixing solution: dilute the 20-fold concentrated washing solution with distilled water to the original one. 3. Set up sample wells: take a sufficient number of enzyme-coated plates and fix them on the frame. Set up standard wells, sample wells to be tested and blank control wells, and record the positions of each well. 4. Add standard product: add 50μL of standard product into the standard well; no blank control well. 5. Add test sample: Add 10 μL of biotin to the test sample well, and then add 50 μL of the test sample; blank control well is not added. 6. Add enzyme-labeled working solution: add 100μL of enzyme-labeled working solution to each well; no blank control well. 7. Incubation: Incubate in a 37 ° C water bath or thermostat for 60 minutes. 8. Wash the plate: discard the liquid, pat dry on the absorbent paper, fill each hole with washing liquid, let stand for 1min, shake off the washing liquid, pat dry on the absorbent paper, repeat the plate washing 4 times (you can also use the washing machine to press Instructions for washing the board). 9. Color development: Add 50μL of developer A solution to each well, then add 50μL of developer B solution, and develop color at 37 ° C in the dark for 15min. 10. Termination: Remove the enzyme labeling plate and add 50μL of stop solution to each well to stop the reaction (the color changes from blue to yellow). 11. Determination: Zero the blank holes, and within 15 minutes after termination, measure the absorbance (OD value) of each well with a wavelength of 450 nm. 12. Calculation: According to the concentration of the standard product and the corresponding OD value, calculate the linear regression equation of the standard curve, and then calculate the corresponding sample concentration on the regression equation according to the OD value of the sample. You can also use various application software to Calculation. The final concentration is the actual measured concentration times the dilution factor. [Sample requirements] 1. The sample must not contain sodium azide (NaN3), because sodium azide (NaN3) is an inhibitor of horseradish peroxidase (HRP). 2. The specimen should be extracted as soon as possible after collection. The extraction should be carried out according to relevant literature. The experiment should be carried out as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided. 3. The sample should be fully centrifuged, without hemolysis and particles. [Notes] 1. The experiment is carried out in strict accordance with the instructions, and the result of the experiment must be determined by the reading of the microplate reader. 2. If the enzyme-labeled coated board is not used up after opening, it should be put into a sealed bag and added with desiccant immediately. 3. It is recommended that all standards, samples and blank controls be tested in duplicate, and the average value is taken to reduce the experimental error. 4. If the color is too light, the substrate incubation time can be extended properly. 5. In order to avoid cross-contamination, the standard, sample and blank control should be replaced with a tip for each additional one; the common components such as enzyme working solution, sample diluent and substrate should be added by cantilever, and should not touch the microwell ; Do not reuse the sealing film. 6. The kits are used within the warranty period, and different batches of reagents should not be mixed. 7. Substrate B is sensitive to light and avoid prolonged exposure to light. [Summary of operating procedures] Prepare reagents, samples and standards Add the prepared samples, standards and enzyme-labeled reagents, incubate at 37 ° C for 60 minutes and wash the plate 4 times, add the developing solutions A and B, add the color at 37 ° C for 15 minutes Calculate the OD value within 15 minutes of the stop solution. [Detection range] 6.25μg / mL-200μg / mL [Specifications] 96T / box [Storage] 2-8 ° C, protected from light and moisture. [Validity period] 6 months
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