ELISA analysis of Î²-secretase activity in rat brain
Kit instruction manual
This kit is for research use only.
Detection range: 96T
1pg / ml-40pg / ml
purpose of usage:
This kit is used to determine the activity of Î²-secretase in the brain of rat serum, plasma and related fluid samples.
This kit uses the double antibody sandwich method to determine the level of Î²-secretase activity in the rat brain in the specimen. Microporous plates were coated with purified rat brain Î²-secretase activity antibody to make solid-phase antibodies, and then the Î²-secretase activity in the brain was added to the monoclonal antibody-coated microwells in turn, followed by HRP labeled The Î²-secretase active 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 Î²-secretase activity in the brain in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the concentration of Î²-secretase in the rat brain in the sample was calculated by a standard curve.
Composition of ELISA kit for Î²-secretase activity in rat brain
30 times concentrated washing solution
20ml Ã— 1 bottle
6ml Ã— 1 bottle
6ml Ã— 1 bottle
Standard product (80pg / ml)
0.5ml Ã— 1 bottle
Enzyme coated plate
12 holes Ã— 8
1.5ml Ã— 1 bottle
6ml Ã— 1 bottle
Developer A liquid
6ml Ã— 1 bottle
Developer B liquid
6ml Ã— 1 / bottle
1. Specimens are extracted as soon as possible after collection, and extraction is performed according to relevant literature, and experiments should be conducted as soon as possible after extraction. If the test cannot be performed immediately, the specimen can be stored at -20 â„ƒ, but repeated freezing and thawing should be avoided
2. The samples containing NaN3 cannot be detected because NaN3 inhibits horseradish peroxidase (HRP) activity.
Operating steps of ELISA kit for Î²-secretase activity in rat brain
1. Dilution of standard products: This kit provides one original standard product. The user can perform dilution in a small test tube according to the following chart.
40pg / ml
Standard No. 5
150Î¼l of original standard is added to 150Î¼l standard dilution
20pg / ml
150Î¼l Standard No. 5 is added to 150Î¼l standard dilution
10pg / ml
150Î¼l Standard No. 4 is added to 150Î¼l Standard Diluent
5pg / ml
Standard No. 2
150Î¼l Standard No. 3 is added to 150Î¼l Standard Diluent
2.5pg / ml
150Î¼l Standard No. 2 is added to 150Î¼l Standard Diluent
2. Sample addition: set up blank wells (the blank control wells do not add samples and enzyme reagents, the rest of the operation is the same), standard wells, sample wells to be tested. Accurately add 50Î¼l of the standard on the enzyme-coated plate, add 40Î¼l of sample diluent to the sample well, 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, shake gently to mix.
3. Incubation: Seal the plate with a sealing plate and incubate at 37 Â° C for 30 minutes.
4. Mixing solution: Dilute 30 times concentrated washing liquid with distilled water 30 times and reserve
5. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with the washing liquid, let it stand for 30 seconds and then discard, repeat 5 times and pat dry.
6. Add enzyme: add 50Î¼l of enzyme label reagent to each well, except blank well.
7. Incubation: The operation is the same as 3.
8. Washing: The operation is the same as 5.
9. Color development: add 50Î¼l of developer A to each well, then add 50Î¼l of developer B, mix gently, and develop at 37 Â° C in the dark for 15 minutes.
10. Termination: Add 50Î¼l of stop solution to each well to stop the reaction (at this time the blue will turn to yellow).
11. Determination: Measure the absorbance (OD value) of each well in sequence with the blank air conditioner at zero and 450 nm wavelength. The measurement should be carried out within 15 minutes after adding the stop solution.
Summary of the operating procedures of the ELISA kit for Î²-secretase activity in the rat brain:
Taking the concentration of the standard as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; Calculate the linear regression equation of the standard curve with the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor to obtain the actual concentration of the sample.
1. The kit should be equilibrated at room temperature for 15-30 minutes before being taken out of the refrigerated environment. If the enzyme label coated plate is unopened, the strip should be stored in a sealed bag.
2. Crystals may be precipitated in the concentrated washing liquid, which can be heated and dissolved in a water bath during dilution, and the results will not be affected during washing.
3. The sampler should be used at each step of sample addition, and the 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.
4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. 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 (Ã— n Ã— 5).
5. The sealing film is limited to one-time use to avoid cross-contamination.
6. Please keep the substrate away from light.
7. Strictly follow the instructions, and the test results must be determined by the microplate reader.
8. All samples, washing liquids and various wastes should be treated as infectious agents.
9. The components of different batches of this reagent shall not be mixed.
10. If there is any difference with the English manual, the English manual shall prevail.
Storage conditions and validity period
1. Kit storage :; 2-8 â„ƒ.
2. Validity: 6 months
Instruction manual of Î²-secretase activity ELISA kit in rat brain
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