TBA Total Bile Acid ELISA Kit

Product attributes:

Required but not provided reagents and equipment:

1. The product is only used for scientific research standard specifications of enzyme-linked immunosorbent assay (ELISA) reader

2. High speed centrifuge

3. Electric constant temperature incubator

4. Clean test tubes and Eppendorf tubes

5. Series adjustable pipettes and suction heads, use multi-channel pipettes when detecting a large number of samples at once

6. Distilled water, volumetric flasks, etc.

Specimen requirements:

1. Extract the specimen as soon as possible after collection, according to relevant literature, and conduct experiments as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be stored at -20 ℃, but repeated freezing and thawing should be avoided.

2. Samples containing NaN3 cannot be detected because NaN3 inhibits the activity of horseradish peroxidase (HRP).

Preparation of reagents and collection of blood samples:

1. Collect specimens: Serum, plasma (EDTA, citrate, heparin anticoagulant), cell culture supernatant, tissue homogenate, etc. should be tested as early as possible and stored at 2-8 ℃ for 48 hours; Longer storage time should be frozen (-20 ℃ or -70 ℃) to avoid repeated freeze-thaw cycles.

2. Standard solution preparation: Add 0.5ml of distilled water and mix well before use to prepare a solution of 1200ng/ml. Set up 8 standard tubes, add 900ul of sample diluent to each tube, and add 500ul of sample diluent to the second to eighth tubes. Add 100ul of standard solution with a concentration of 1200ng/ml into the tube and mix well. Use a sampler to aspirate 500ul and transfer it to the second tube. Repeat the process of diluting by multiples and remove 500ul from the seventh tube. The eighth tube is a blank control.

3. Dilute 10 times the sample diluent with distilled water at a ratio of 1:10 (Example: 1ml concentrated diluent+9ml distilled water).

4. Washing solution: Dilute with distilled water at a ratio of 1:20 (Example: 1ml of concentrated washing solution added to 19ml of distilled water)

Detection program:

1. Sample addition: Add 100ul of standard or test sample to each well, mix the reaction plate thoroughly, and place it at 37 ℃ for 120 minutes.

2. Plate washing: Wash the reaction plate thoroughly 4-6 times with washing solution and print it dry on filter paper.

3. Add 100ul of antibody working solution to each well. Mix the reaction plate thoroughly and place it at 37 ℃ for 60 minutes.

4. Board washing: Same as before.

5. Add 100ul of enzyme labeled antibody working solution to each well. Place the reaction plate at 37 ℃ for 30 minutes.

Preparation before sample experiment:

ELISA imported reagent kit liquid samples: including serum, plasma, urine, pleural and peritoneal fluid, cerebrospinal fluid, cell culture supernatant, etc.

(1) Serum

After natural coagulation of blood at room temperature for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 revolutions per minute). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(2) Plasma:

EDTA, sodium citrate, or heparin should be selected as anticoagulants according to the requirements of the specimen. After mixing for 10-20 minutes, centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(3) Urine:

Collect with sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again. Follow this procedure for pleural effusion, ascites, and cerebrospinal fluid.

(4) Cell culture supernatant:

When detecting secretory components, collect them using sterile tubes. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant.

(5) Cultivate cells

When detecting intracellular components, dilute the cell suspension with PBS (pH 7.2-7.4) to a cell concentration of around 1 million/ml. By repeatedly freezing and thawing or adding tissue protein extraction reagents, cells are destroyed and intracellular components are released. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. If precipitation forms during storage, it should be centrifuged again.

(6) Organizational specimen

After cutting the specimen, weigh it. Add a certain amount of PBS, pH 7.4. Quickly freeze and store in liquid nitrogen for future use. The specimen remains at a temperature of 2-8 ℃ even after melting. Add a certain amount of PBS (pH 7.4) or tissue protein extraction reagent, and homogenize the specimen manually or with a homogenizer. Centrifuge for about 20 minutes (2000-3000 rpm). Carefully collect the supernatant. After packaging, one portion is to be tested, and the rest is to be frozen for future use.

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Phosphate Acetyl Coenzyme A Carboxylase Alpha (Ser78) Phosphorylated Acetyl Carboxylase Antibody

Phosphoro-Ack1 (Tyr859+860) phosphorylated Ack1 antibody

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Operation steps:

1. Dilution of standard samples: This kit provides one original standard sample, and users can dilute it in a small test tube according to the following chart.

2. Sample addition: Set up blank wells (blank control wells do not contain samples or enzyme-linked immunosorbent assay reagents, all other steps are the same), standard wells, and wells for the test sample. Accurately add 50 μ l of the standard sample on the enzyme-linked immunosorbent assay (ELISA) coated plate, first add 40 μ l of sample diluent to the well of the test sample, and then add 10 μ l of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding touching the well wall as much as possible, and gently shake and mix well.

3. Incubation: Cover the plate with a sealing film and incubate at 37 ℃ for 30 minutes.

4. Liquid preparation: Dilute 30 times the concentrated washing solution with distilled water and set aside for later use.

5. Washing: Carefully remove the sealing film, discard the liquid, shake dry, fill each hole with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times and pat dry.

6. Enzyme addition: Add 50 μ l of enzyme labeled reagent to each well, except for blank wells.

7. Incubation: Follow the same procedure as in 3.

8. Washing: Follow the same procedure as in 5.

9. Color development: Add 50 μ l of color developing agent A50 to each well, then add 50 μ l of color developing agent B50, gently shake and mix, and develop color at 37 ℃ in the dark for 15 minutes.

10. Termination: Add 50 μ l of termination solution to each well to terminate the reaction (at this point, blue will turn yellow).

11. Measurement: Measure the absorbance (OD value) of each well in sequence using blank air conditioner zero and 450nm wavelength. The measurement should be conducted within 15 minutes after adding the termination solution.