Rat cyclic adenosine phosphate (cAMP) ELISA work guide
This reagent is for research purpose only: This kit is used to determine the content of cAMP in rat serum, plasma and related liquid samples.
Experimental principle:
This kit uses the double antibody sandwich method to determine the level of cAMP in rats. Microporous plates were coated with purified rat cyclic adenosine monophosphate (cAMP) antibody to prepare solid-phase antibodies, and cyclic adenosine monophosphate (cAMP) was added to the microwells coated with mAb in turn, followed by HRP-labeled cyclic phosphate Adenosine (cAMP) 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 cAMP in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of rat cyclic adenosine phosphate (cAMP) in the sample was calculated by a standard curve.
Sample processing and requirements:
1. 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 ° 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.
2. 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.
3. Serum: blood coagulates naturally at room temperature 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.
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. Plasma: EDTA, sodium citrate or heparin 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.
Precautions:
a. The kit should be taken out of the refrigerated environment and should be equilibrated at room temperature for 15-30 minutes before use. If the enzyme label coated plate is unopened after opening, the strip should be stored in a sealed bag.
B. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in the water bath during dilution, and the result will not be affected during washing.
C. The sample adder should be used at each step of sample addition, and its accuracy should be regularly checked to avoid test errors. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
D. Please make a standard curve at the same time of each measurement, it is best to make multiple holes. 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 well), please first dilute it with a certain multiple (n times) of the sample diluent and then determine it. When calculating, please multiply the total dilution Multiple (× n × 5).
E. The sealing film is only for one-time use to avoid cross contamination.
If you have any questions, please contact: Shanghai Yanjin Biological Technology Co., Ltd.!
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