Alkaline phosphatase activity colorimetric assay kit. The phosphatase assay kit is designed to measure the activity of phosphatases in biological samples and to screen for agonists and inhibitors of phosphatases. Elevated alkaline phosphatase and its interpretation dr kasi md duration. Faster reaction setup no supplemental additives like zinc required flexible reaction conditions active in any restriction enzyme buffer, no cleanup required. However, some colorimetric assays allow quantification of the hydrolyzed product without interference with the sample. The sub strate chosen was the wellstudied paranitrophenylphosp hate pnpp substrate which.
The phosphatase assay kit uses paranitrophenyl phosphate pnpp, a chromogenic substrate for most phosphatases, including alkaline. A very robust and inexpensive kinetic assay for determining rates of hydrolysis of p. Sensolyte pnpp alkaline phosphatase assay kit colorimetric. Assay kits, phosphatase assay kits, sensolyte pnpp alkaline phosphatase assay kit colorimetric.
The alkaline phosphatase enzyme at each dilution was mixed with pnpp substrate, and then incubated at room. Identification of staphylococcus pyogenes by the phosphatase reaction. Alkaline phosphatase, calf intestinal cip nonspecifically catalyzes the dephosphorylation of 5. Detection of alkaline phosphatase with the sensolyte pnpp alkaline phosphatase assay kit. Alkaline phosphatase assay information alkaline phosphatase assay kit colorimetric is designed to measure alp activity directly in biological samples without pretreatment.
Phosphatase, alkaline from bovine intestinal mucosa was serially diluted in assay bufferalkaline, and its activity was measured with sciencell pnpp phosphatase assay after a given time of reaction 15 min and. Alkaline phosphatase assay kit colorimetric ab83369 is a highly sensitive, simple, direct and htsready colorimetric assay designed to measure alkaline phosphatase alp activity in serum and other mammalian samples. Colorimetric analysis of alkaline phosphatase activity in s. Bioassay systems quantichrom alkaline phosphatase assay kit is designed to measure alp activity directly in biological samples without pretreatment. Alkaline phosphatase alp, alkp, alpase, alk phos ec 3. Alkaline phosphatase alp catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. L incubate at rt or 37c for 530 min read absorbance at 400 nm. Quantichromtm alkaline phosphatase assay kit dalp250 colorimetric kinetic determination of serum alkaline phosphatase activity description alkaline phosphatase alp catalyzes the hydrolysis of phosphate esters in an alkaline environment, resulting in the formation of an organic radical and inorganic phosphate. Changes in alkaline phosphatase level and activity are associated with various disease states in the liver and bone.
Enzymatic assay of alkaline phosphatase, diethanolamine assay. Pnpp p nitrophenyl phosphate, disodium salt is a widely used substrate for detecting alkaline phosphatase in elisa applications. The reaction yields paranitrophenol, which becomes an intense yellow soluble product under alkaline conditions and can be. Shrimp alkaline phosphatase rsap is a heat labile alkaline phosphatase purified from a recombinant source. Inhibitors of alp, such as edta, oxalate, fluoride, and citrate should be avoided in sample preparation. Ensure that the assay buffer is at room temperature before use. Prepare enough pnpp substrate by mixing per assay 0. Try the sigma alkaline phosphatase yellow pnpp liquid substrate system for elisa. Our phosphatase substrate kit contains pnpp tablets and a diethanolamine buffer to yield more than one liter of substrate. Is anyone are familiar with alp assay for bone regeneration osteoblast. Like all enzymes, it is needed in small amounts to trigger specific chemical reactions. Phosphatase, alkaline from bovine intestinal mucosa was serially diluted in assay bufferalkaline, and. The kit provides all the essential components with our optimized mix and read assay protocol that is. In this protocol, we describe one of the most commonly used ptp assays that.
The pnpp phosphatase activity is measured using a continuous or singlepoint spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of pnpp to pnitrophenol, a chromogenic product with absorbance at 405 nm 1. L add alkaline phosphatase standards or test samples 50. Alkaline phosphatase assay kit is a highly sensitive, simple, direct and htsready colorimetric assay designed to measure alp activity in serum and biological samples. Amplite colorimetric alkaline phosphatase assay kit yellow. Alkaline phosphatase activity colorimetric assay kit k412. Keep samples, alp enzyme and pnpp solution on ice during the assay. It is the responsibility of all trained analytical services personnel to follow this protocol as written. For example, hydrolysis of pnpp, which by itself is colorless, generates p nitrophenol, which is bright yellow at an alkaline ph and whose absorbance can be measured at 405 nm. Overview alkaline phosphatase assay kit colorimetric ab83369 is a highly sensitive, simple, direct and htsready colorimetric assay designed to measure alkaline phosphatase alp activity in serum and other biological samples. The reaction yields paranitrophenol, which becomes an intense yellow soluble product. Each monomer contains five cysteine residues, two zinc atoms and one magnesium atom crucial to its catalytic function, and it is optimally active at alkaline ph environments. The use of bacterial alkaline phosphatase assay for rapid monitoring of bacterial counts on spinach. When alkaline phosphatase and pnpp are reacted, a yellow watersoluble reaction product is formed.
Equilibrate all reagents to room temperature by allowing them to stand for 30 minutes at room temperature. The sensolyte pnpp alkaline phosphatase assay kit provides a convenient colorimetric assay for detecting alkaline phosphatase in biological samples and in elisa with. This product is a readytouse 1x proprietary buffered solution of pnpp. The alkaline phosphatase assay kit is a highly sensitive. The system allows the use of a variety of buffer conditions and substrates, including naturally phosphorylated proteins or synthetic phosphopeptides.
Highthroughput ready colorimetric assay to measure alkaline phosphatase alp activity in cells, serum, plasma etc. Kinetic studies with alkaline phosphatase in the presence and. Alkaline phosphatase is an enzyme that is found in the blood, intestine, liver, and bone cells of the human body. The enzyme is competitively inhibited by the reaction products, uncompetitively inhibited by l. The improved method utilizes pnitrophenyl phosphate that is hydrolyzed by alp into a yellow colored product maximal absorbance at 405nm. The alkaline phosphatase assay kit is a highly sensitive, simple, direct and hts. However, high levels of this enzyme in the body may be due. P1146 and alkaline from bovine intestinal mucosa sigma cat.
Colorimetric alkaline phosphatase assay kit, yellow color. Alkaline phosphatase alp activity assay kit video biovision, inc. Phosphatase, acid from potato was serially diluted in assay bufferacidic, and its activity was measured with sciencell pnpp phosphatase assay after a given time of reaction 15 min and 45 min. Assay protocol assay procedure procedure using 96well plate. Ftjq6720 directions for use protocol for one 96well plate brief summary prepare assay reaction mixture 50. This amplite alkaline phosphatase assay kit uses pnpp, a chromogenic phosphatase substrate, to quantify alkaline phosphatase activity in solutions, in cell extracts as well as on solid surfaces such as pvdf membranes. This assay is widely used for detection of alkaline phosphatase activity in elisa, but can be used in many instances where protein phosphatases are assayed. The pnpp phosphatase activity is measured using a continuous or singlepoint spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of pnpp to p nitrophenol, a chromogenic product with absorbance at 405 nm 1. P6772 were serially diluted in assay bufferacidic and. They can be used to measure alkaline phosphatase, neutral phosphatase, and acid phosphatase activity. Thermo scientific pierce pnpp pnitrophenyl phosphate is a colorimetric, soluble substrate of alkaline phosphatase for use in elisa applications. Can anyone please recommend a protocol for acid phosphatase enzyme kinetic study. Dephosphorylation of proteins alkaline phosphatase ap is a nonspecific phosphomonoester hydrolase that catalyzes the hydrolysis of a wide variety of organic monophosphates. The change of alkaline phosphatase activity is involved in a variety of physiological and pathological events, such as bone development, bonerelated diseases, gestationrelated diseases and inflammatory bowel diseases.
The reaction increases in rate with increase in ph. Alkaline phosphatase assay kit bioassay systems 8777823888. Enzymatic assay of alkaline phosphatase, diethanolamine. A protocol for fast, convenient measuring of protein serinethreonine phosphatase activity. The rate of the reaction is directly proportional to the enzyme activity. The sensolyte pnpp alkaline phosphatase assay kit is used in elisa which alkaline phosphatase ap conjugates, apantibody or apstreptavidin, are served as the secondary detection system. K412 alkaline phosphatase activity colorimetric assay kit. Sep 11, 2014 this video demonstrates how to perform a colorimetric alkaline phosphatase activity assay in biological samples using a simple, highly sensitive, addincubateread protocol. Alkaline phosphatase yellow pnpp liquid substrate system for elisa ready to use solution. I am using saos2 osteosarcoma and i am looking for anonkit protocol for measuring alp alkaline phosphatase. Basic protocol 1 colorimetric assay of protein phosphatases using pnitrophenylphosphate. Alkaline phosphatase assay kit colorimetric is designed to measure alp activity directly in biological samples without pretreatment. Alkaline phosphatase yellow pnpp liquid substrate system. Sensolyte pnpp alkaline phosphatase elisa assay kit.