["Collaborating Expert | M.S. Hanyin Sun", "Biology and Medicine University of Chinese Academy of Sciences"], ["Reviewed by | Dr. Yueh-Lin Huang", "Physiology Dalian Medical University"]

Summary

Acid phosphatase (ACP) is widely found in various cells, and is often abnormally expressed in some cancer cells. Sodium p-nitrophenol phosphate is used as the substrate of ACP, which can be hydrolyzed under alkaline conditions to produce yellow p-nitrophenol, and ACP can be quantitatively analyzed by colorimetric assay.

Appliance

Used to determine the level of ACP in tissues or cells.

Operation method

Sodium p-nitrophenol phosphate method

Materials and Instruments

Tissue or cells, RIPA (weak to medium) lysate, sodium p-nitrophenol phosphate solution, buffers
Spectrophotometer, thermostatic water bath, scissors, mortar and pestle, test tubes, etc.

Move

1. take the cell sediment or tissue, add RIPA lysis solution, use scissors or a mortar to fully lyse the cells or tissue (on ice), collect the lysate into a centrifuge tube and centrifuge for 15 min at 15,000 rpm at 4 °C. collect the supernatant, which now contains acid phosphatase.

2. Add buffer and sodium p-nitrophenol phosphate solution. Take a small amount of the supernatant and dilute it with _ddH2O (about 5-10 times). Add an equal amount of buffer and sodium p-nitrophenol phosphate solution to the negative control group (acid phosphatase-free group) and the enzyme activity group. After mixing, the reaction was held at 37 °C in a constant temperature water bath for 10~15 min with shaking every 5 min, and at the end of the reaction, the reaction was terminated by adding 0.5 M NaOH solution. At this time, pH = 14, take 1 mL of liquid at 400 nm spectrophotometer to test the absorbance.

3. Enzyme activity (V: volume of enzyme, V1: volume of enzyme used each time)

Caveat

1. The reaction should be carried out on ice and kept at a low temperature throughout.

2. the extracted enzyme should be reacted immediately and stored at -20 °C and tested as soon as possible.

3. p-Nitrophenol appears yellow when NaOH is added.

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