Trace Method for Coenzyme I NAD (H) Content Test Kit

This reagent kit is for research purposes only. The company's laboratory provides free proxy testing, and the results will be available after 7 working days. The original data is provided!

Product Introduction:

Reagent composition and preparation:

Extraction solution: Liquid 100 mL x 1 bottle, stored at 4 ℃.

Reagent 1: Liquid 5 mL x 1 bottle, stored at 4 ℃ in the dark.

Reagent 2: Liquid 15 mL x 1 bottle, stored at 4 ℃.

Reagent 3: Liquid 3 mL x 1 bottle, stored at 4 ℃.

Reagent 4: Liquid 3 mL x 1 bottle, stored at 4 ℃.

The experimental results indicate that I have a clever trick:
1. Quantitative determination results are calculated based on the standard curve to determine the content of the analyte in the sample.
2. The threshold for positive results is set as the OD value of the sample well being greater than the negative control OD value by+2-3SD.
3. A P/N value (positive pore OD value/negative pore OD value) greater than or equal to 2.1 is considered positive, a P/N value less than 2.1 but greater than 1.5 is suspicious, and a P/N value less than 1.5 is negative.
4. ELISA assay kit quantitative method: After adding substrate, enzymatic hydrolysis and termination of reaction, the color of the positive well is significantly darker than (the competition rule is lighter than) the negative control by naked eye observation. If the color is close to that of the negative control, it is judged as negative.
Sharing with you the classification and requirements of commonly used unstable reagents:
① Volatile and low ignition point reagents should be sealed and stored in a cool, ventilated, and away from sources of fire.
② Substances that react with water vapor and water should be sealed and stored away from water sources.
③ Reagents that are prone to react with oxygen should be stored in solid or crystalline form and should not be stored in aqueous solutions for a long time. Sulfuric acid and hydrogen sulfate solutions should be stored in sealed form, and potassium, sodium, and white phosphorus should be stored in liquid sealed form.
④ Substances that react with carbon dioxide should be sealed and stored, as their corresponding solutions are more reactive than solids, so it is even more important to pay attention to sealed storage.
⑤ Volatile or self decomposing reagents should be sealed and stored in a cool and ventilated place.

Operation steps:

1. Sample preparation:

a. Preparation of cell samples: Collect cells and wash 1-2 times with PBS or physiological saline pre cooled at 4 ℃ or in an ice bath. Pre cooled tissue cell lysate at 4 ℃ or ice bath homogenate for precipitation (glass homogenizer or various common electric homogenizers can be used). Subsequently, centrifuge the homogenate at 4 ℃ and take the supernatant as the sample to be tested.

b. Preparation of tissue samples: Animals were perfused with physiological saline (0.9% NaCl, containing 0.16mg/ml) to remove blood and obtain tissue samples. Add 500-1000ul of tissue cell lysate per 100mg, homogenize at 4 ℃ or in an ice bath (glass homogenizer or various common electric homogenizers can be used). Subsequently, centrifuge the homogenate at 4 ℃ and take the supernatant as the sample to be tested.

d. Measure protein concentration using BCA protein concentration assay kit (P1511). Usually, the average SOD activity in cell or tissue homogenate samples with 10-20 μ g protein is about 1 activity unit (the differences between different cells and tissues may be significant, and this activity range is only used as a preliminary reference). Preparing 20-100 ug of protein for each sample is usually sufficient for subsequent testing.

e. Dilute the sample appropriately with the SOD detection buffer provided by the kit based on the protein concentration and expected protein usage. For example, mouse liver typically needs to be diluted 10-100 times. If the prepared samples are measured on the same day, they can be stored in an ice bath; If the measurement cannot be completed on the same day, it can be frozen at -70 ℃, but it is recommended to complete the measurement on the same day as much as possible.

2. Preparation of reagent kit:

a. Preparation of WST-8 enzyme working solution: Refer to the table below and prepare an appropriate amount of WST-8 enzyme working solution based on the number of samples (including standard samples) to be tested. The prepared WST-8 enzyme working solution should be stored at 4 ℃ or in an ice bath and can be used on the same day, but it is recommended to prepare and use it as soon as possible.

Attention: Due to the small amount and easy settling of enzyme solution, it is necessary to gently centrifuge it before use, and then mix it properly before use.

b. Preparation of reaction initiation working solution: Dissolve the reaction initiation solution (40X) provided by the reagent kit, mix well, and dilute it by adding 1 μ l of reaction initiation solution (40X) to 39 μ l of SOD detection buffer. This is the reaction initiation working solution. Stored at 4 ℃ or in an ice bath, it can be used on the same day, but it is recommended to prepare and use it as soon as possible.

c. (Optional) Preparation of SOD standard: Self prepared SOD standard is required. Dilute the SOD standard with the diluent provided in this kit to the following series of concentrations: 20U/ml, 10U/ml, 5U/ml, 2.5U/ml, 1.25U/ml, 0.625U/ml. In the subsequent testing, 20 microliters can be taken each as a reference sample for testing. Explanation: To avoid a decrease in SOD enzyme activity after dilution, SOD standard should be diluted and used immediately; This kit does not require SOD as a standard for SOD detection, but SOD standard can be used as a positive control or as a reference for quantifying SOD activity.

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C-type lectin domain family 2 member C (CLEC2C) recombinant protein Recombinant C-type lectin domain family 2, Member C (CLEC2C)

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Discs Large Homologg Associated Protein 5 (DLGAP5) recombinant protein Recombinant Discs

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Recombinant GATA Binding Protein 3 (GATA3)

Recombinant GATA Binding Protein 4 (GATA4)

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