Human high molecular weight cytokeratin (CK-HMW) enzyme-free ELISA instructions This reagent is for research use only: this kit is used to determine the content of high molecular weight cytokeratin (CK-HMW) in human serum, plasma and related liquid samples. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human high molecular weight cytokeratin (CK-HMW) in the specimen. Microporous plates were coated with purified human high molecular weight cytokeratin (CK-HMW) antibody to make solid-phase antibodies, and high molecular weight cytokeratin (CK-HMW) was added to the monoclonal antibody-coated microwells in turn, followed by HRP-labeled high molecular weight cytokeratin (CK-HMW) antibody binds to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and into the final yellow under the action of acid. The color depth is positively correlated with the high molecular weight cytokeratin (CK-HMW) in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of human high molecular weight cytokeratin (CK-HMW) in the sample was calculated by a standard curve. Kit composition: kit composition 48 well configuration 96 well configuration storage instructions 1 copy 1 copy sealing film 2 pieces (48) 2 pieces (96) 1 sealed bag 1 enzyme coated plate 1 Ã— 48 1 Ã— 96 2 Store standard at -8 Â° C: 18ng / ml 0.5ml Ã— 1 bottle 0.5ml Ã— 1 bottle at 2-8 Â° C Store standard dilution 1.5ml Ã— 1 bottle 1.5ml Ã— 1 bottle at 2-8 Â° C Store enzyme reagent 3 ml Ã— 1 bottle of 6 ml Ã— 1 bottle of sample diluent at 2-8 Â° C 3 ml Ã— 1 bottle of 6 ml Ã— 1 bottle of 2-8 Â° C Developer A 3 ml Ã— 1 bottle of 6 ml Ã— 1 bottle of 2-8 Â° C Developer B solution 3 ml Ã— 1 bottle 6 ml Ã— 1 bottle at 2-8 Â° C storage stop solution 3ml Ã— 1 bottle 6ml Ã— 1 bottle at 2-8 Â° C Concentrated washing solution (20ml Ã— 20 times) Ã— 1 bottle (20ml Ã— 30 times) Ã— 1 bottle at 2-8 Â° C to store the sample. Processing and requirements: 1. Serum: room temperature blood is naturally coagulated for 10-20 minutes, centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully and centrifuge again if a precipitate appears during storage. 2. Plasma: EDTA or sodium citrate should be selected as the anticoagulant according to the requirements of the specimen, mixed for 10-20 minutes, and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 3. Urine: collected in a sterile tube and centrifuged for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, centrifuge again. Pleural and ascites, cerebrospinal fluid reference implementation. 4. Cell culture supernatant: When detecting secreted components, collect with a sterile tube. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. When detecting the components inside the cells, dilute the cell suspension with PBS (PH7.2-7.4), and the cell concentration will reach about 1 million / ml. Through repeated freezing and thawing, the cells are destroyed and the intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. If a precipitate forms during storage, it should be centrifuged again. 5. Organize the specimen: after cutting the specimen, weigh it. Add a certain amount of PBS, PH7.4. Quickly freeze and save with liquid nitrogen for later use. After the specimen melts, it still maintains a temperature of 2-8 Â° C. Add a certain amount of PBS (PH7.4) and homogenize the specimen with a manual or homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Collect the supernatant carefully. After aliquoting, a portion is to be tested, and the rest is frozen for future use. 6. 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 Â° C, but repeated freezing and thawing should be avoided. 7. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP). Operating steps Dilution and sample loading of standards: set up 10 wells of the standard on the enzyme-coated plate, add 100 Î¼l of the standard in the first and second wells, and then add the standard in the first and second wells 50Î¼l of diluent, mix well; then take 100Î¼l from the first well and the second well and add them to the third and fourth wells respectively, and then add 50Î¼l of standard diluent to the third and fourth wells respectively, mix well; Then take 50Î¼l each in the third and fourth wells and discard it, then add 50Î¼l each to the fifth and sixth wells, and then add 50ul of the standard dilution solution to the fifth and sixth wells respectively, and mix well; After mixing, take 50Î¼l from the fifth and sixth wells and add them to the seventh and eighth wells respectively. Then add 50Î¼l of the standard dilution solution to the seventh and eighth wells respectively. Take 50Î¼l from the eight wells and add them to the ninth and tenth wells. Then add 50Î¼l of the standard dilution solution to the ninth and tenth wells. After mixing, take 50Î¼l from the ninth and tenth wells and discard. (After dilution, the volume of each well is 50Î¼l, and the concentrations are 12 ng / ml, 8 ng / ml, 4 ng / ml, 2 ng / ml, and 1 ng / ml. Sample addition: set blank wells (blank control wells are not Add the sample and the enzyme reagent, the rest of the steps are the same), the sample well to be tested. Add 40Î¼l of the sample diluent to the sample well of the enzyme-coated plate, and then add 10Î¼l of the sample to be tested (the final dilution of the sample is 5 Times). Add the sample and add the sample to the bottom of the well of the microplate, try not to touch the wall of the well, gently shake to mix. Incubation: seal the plate with the sealing plate and incubate at 37 â„ƒ for 30 minutes. Dosing solution: add 20 The concentrated washing solution was diluted 20 times with distilled water before use. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with the washing solution, let it stand for 30 seconds, then discard, repeat 5 times, pat dry Add enzyme: add 50Î¼l of enzyme labeling reagent to each well, except for blank wells. Incubate: operate the same as 3. Wash: operate the same as 5. Color development: first add the developer A50Î¼l to each well, then add the developer B50Î¼l, gently Mix by shaking and develop for 15 minutes in the dark at 37 Â° C. Termination: Add 50Î¼l of stop solution to each well to stop the reaction (blue to yellow at this time) .Determination: The absorbance (OD value) of each well is measured in sequence with a blank air conditioner at zero, 450nm wavelength. The determination should be performed within 15 minutes after adding the stop solution. Note: The kit should be taken out of the refrigerated environment and equilibrated at room temperature 15- It can be used after 30 minutes. If the enzyme-coated plate is not used up after opening, the slats should be stored in a sealed bag. The concentrated washing liquid may crystallize out. When diluted, it can be heated and dissolved in a water bath. Does not affect the results. The sampler should be used at each step of sample addition, and its accuracy should be regularly checked to avoid test errors. The time of one sample injection is best controlled within 5 minutes. If there are many specimens, it is recommended to use a rifle to add samples. Please make a standard curve at the same time of each measurement, preferably a double well. If the content of the test substance in the specimen is too high (the OD value of the sample is greater than the OD value of the first well of the standard product), please dilute it with a certain multiple of the sample diluent first (N times) after the measurement, please multiply the total dilution factor (Ã— n Ã— 5) at the end of the calculation. The sealing film is only for one-time use to avoid cross-contamination. The substrate should be kept away from light. Strictly follow the instructions Carry on If the determination must be based on the reading of the microplate reader. All samples, washing solutions and various wastes should be treated as infectious agents. The components of different batches of this reagent should not be mixed. 10. If there are differences from the English instructions, the English instructions shall be used. Calculate: Take the concentration of the standard as the abscissa and the OD as the ordinate, draw a standard curve on the graph paper, and find out the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; or use Calculate the linear regression equation of the standard curve with the concentration and OD value of the standard. Substitute the OD value of the sample into the equation to calculate the sample concentration and multiply it by the dilution factor to obtain the actual concentration of the sample. (This figure is for reference only) Reagent Box performance: 1. The linear regression of the sample and the expected concentration correlation coefficient R value is above 0.92. 2. The batch and batch see should be less than 9% and 15% respectively. Storage conditions and expiration date: 1. Kit storage :; 2-8 â„ƒ. 2. Validity: 6 months
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