Background[1-7]
The acid phosphatase detection kit is used to quickly and conveniently detect endogenous acid phosphate in the supernatant, plasma, serum, urine and other samples of lysis or homogenate products of cell or tissue samples. Enzyme activity kit. Acid Phosphatase, also known as acid phosphatase, is an acid hydrolase with a high content in lysosomes and is considered a marker for identifying subcellular components of lysosomes. Acid phosphatase is a family of proteins with molecular weights ranging from 18kD to 100kD in mammals. Acid phosphatase is divided into two categories, one is tartrate-sensitive type and the other is fluoride ion-sensitive type. Acid phosphatase in lysosomes is tartrate-sensitive, whereas acid phosphatase in erythrocytes and macrophages is fluoride-sensitive.
The activity range of acid phosphatase in plasma is 2-7.9U/L, and the activity range of acid phosphatase in serum is 2.5-11.7U/L. Semen contains a high concentration of acid phosphatase, and its activity can reach 87-436KU/L. Acid phosphatase can remove the phosphate group of phosphate esters under acidic conditions. There are many acid phosphatase isoenzymes in the human body, and their concentrations in serum can be used as important parameters for diagnosing diseases of corresponding organs. Prostatic acid phosphate Excessive enzyme levels may indicate tumors in the prostate, while high levels of tartrate-resistant acid phosphatase may indicate the risk of bone cancer. Therefore, it is of great significance to detect the activity of acid phosphatase in corresponding tissue cells.
Detection principle: Para-nitrophenyl phosphate (pNPP) is a commonly used phosphatase chromogenic substrate. Under acidic conditions, para-nitrophenol can be generated under the action of acid phosphatase. Para-nitrophenol (p-nitrophenol) appears as a yellow product under alkaline conditions, and the absorbance can be detected at -415nm. The darker the yellow color of the product, the higher the acid phosphatase activity, and vice versa. From this, the acid phosphatase activity level can be calculated through colorimetric analysis.
Notes:
1. If you want to perform absolute quantification of enzyme activity, you must pay attention to precise timing when performing enzyme reactions. At this time, it is recommended to use a longer incubation time such as 30 minutes to reduce the time error during the operation. At the same time, if the enzyme activity in the sample is high, the sample can be appropriately diluted in advance.
2. Various acid phosphatase inhibitors must be avoided in the sample solution.
3. One tube of chromogenic substrate needs to be used on the same day after preparation, so please be careful to prepare more samples for testing together to avoid wastage of the kit.
4.p-nitrophenol solution is harmful to the human body. Please be careful when handling it and pay attention to effective protection to avoid direct contact with the human body or inhalation. The reaction stop solution is corrosive, so please be careful when handling it and pay attention to effective protection to avoid direct contact with the human body or corrosion of other items.
Apply[8-9]
The acid phosphatase detection kit can be used to detect the proliferation status of tumor cells using the acid phosphatase method
Based on the close relationship between acid phosphatase activity in the cytoplasm and cell proliferation, the correlation between cell number and acid phosphatase activity was determined, a 96-well plate micro-assay method was established, and flow cytometry was used at the same time Detect cell cycle and apoptosis, determine the relationship between apoptotic cells and acid phosphatase, and compare the sensitivity with the thiazole blue (MTT) method. Results In two liver cancer cell lines (HepG2 cells and CBRH 7919 cells), the activity of acid phosphatase was directly proportional to the number of cells, and there was a linear relationship within the range of (0.5-7) × 10 3 cell number. The correlation coefficient between the two (CV) reached 0.994; 1 hour after phorbol ester (TPA) stimulated liver cancer cells, the activity of acid phosphatase increased significantly.
On the contrary, after the action of arsenic trioxide, the activity of acid phosphatase decreases significantly. The higher the concentration, the more significant the decrease. After arsenic trioxide acted on HepG2 cells for 2 and 4 hours, apoptotic cells accounted for only 3.98%. On CBRH 7919 cells, the activity of acid phosphatase decreased significantly before apoptosis occurred. Conclusion Detecting the acid phosphatase activity of cells can be used as an indicator of cell proliferation and apoptosis. This method is simple and rapid, and can be used for screening large quantities of anti-tumor drugs.
References
[1]Interdependent regulation of intracelluler acidification and SHP-I in apoptosis.Thangaraju M,Sharma K,Liu D,et al.Cancer Research.1999
[2]An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells.Yang TT,Sinai R,Kain SR.Analytical Biochemistry.[3]1996A novel alkalineα-galactosidase gene is involved in rice leaf senescence[J] .Ruey-Hua Lee,Mei-Chung Lin,Shu-Chen Chen.Plant Molecular Biology.2004(2)
[4]Expression of anα-galactosidase from Saccharomyces cerevisiae in Aspergillus awamori and Aspergillus oryzae[J].R-A Murphy,R-F-G Power.Journal of Industrial Microbiology&Biotechnology.2002(2)
[5]Multipleα-galactosidases from Aspergillus niger: purification, characterization and substrate specificities[J].Enzyme and Microbial Technology.2001(6)
[6]A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase andα-galactosidase[J].J.H.Clarke,K.Davidson,J.E.Rixon,J.R.Halstead,M.P.Fransen,H.J.Gilbert,G.P. Hazlewood.Applied Microbiology and Biotechnology.2000(6)
[7]Cloning and expression of a member of the Aspergillus niger gene family encodingα-galactosidase[J].Irma F.Herder,Ana M.Mateo Rosell,Caren M.Zuilen,Peter J.Punt,Cees A.M.J.J.Hondel. MGG Molecular&General Genetics.1992(3)
[8]Induction and genetics of twoα-galactosidase activitie antioxidants in Saccharomyces cerevisiae[J].Richard G.Buckholz,Bruce G.Adams.MGG Molecular&General Genetics.1981(1)
[9]Lu Hong, Wu Xingzhong. Detection of tumor cell proliferation status by acid phosphatase method[J]. Chinese Journal of Laboratory Medicine, 2001(02):19-21.
