Is pNPP a good substrate?
Uptima pNPP generally outperforms other available AP substrates in most applications, and often similar chromogenic detection systems with peroxidase. Stability The substrate is stable for more than 30 months at +4°C.
How do you do ALP assay?
Assay Procedure To measure intracellular ALP, washed cells (1×105) can be homogenized in 50 μl Assay Buffer, centrifuge to remove insoluble material at 13,000g for 3 minutes. Add different volume of samples into 96-well plate; bring the total volume to 80 µl with Assay Buffer. Colored samples may interfere with O.D.
What are the methods used for acid phosphatase determination?
Acid phosphatase activity is determined by splitting 1-naphthyl phosphate, concurrently diazotizing the released 1-naphthol with Fast Red TR, and measuring the resulting color. The test is performed in the presence and absence of tartrate.
Why PNPP is used as a substrate?
PNPP (p-Nitrophenyl Phosphate, Disodium Salt) is a widely used substrate for detecting alkaline phosphatase in ELISA applications. When alkaline phosphatase and PNPP are reacted, a yellow water-soluble reaction product is formed. This reaction product absorbs light at 405nm. The substrate is offered in several formats.
What Colour is PNPP?
pNPP (p-nitrophenylphosphate) is a chromogenic substrate for Alkaline Phosphatase (AP). In the presence of AP, pNPP is hydrolyzed rapidly to p-nitrophenol and inorganic phosphate. Liberated p-nitrophenol is seen as a clear yellow color that is measured at 405/620 nm.
How did you calculate the final concentration of pNPP?
Molecular Weight of PNPP is 461.4 daltons. The concentration in 2.3 mL will be 80.28/2.3 = 34.9 µg/mL or 34.9 mg/L or 0.349% (w/v) or 34.9/401.4 = 0.0756 mM.
Is pNPP yellow?
What is the principle of alkaline phosphatase test?
This test is based on the principle that the alkaline phosphatase enzyme in raw milk liberates phenol from a disodium para-nitro phenyl phosphate and forms a yellow coloured complex at alkaline pH (Scharer, 1943). The intensity of yellow colour produced is proportional to the activity of the enzyme.
How do you prepare alkaline phosphatase?
Prepare diazonium salt solution: Dissolve contents of one Fast Blue RR Salt capsule or one Fast Violet B capsule in distilled water (from step 1). A magnetic stirrer may be helpful. 3. Add 2 ml Naphthol AS-MX Phosphate Alkaline Solution to diluted diazonium salt solution (from step 2).
What Colour is pNPP?
Why pNPP is used as a substrate?
How do you calculate absorbance from Vo?
To do this, you calculate the slope of the linear standard curve, which is in units of absorbance change/µM PNp. Divide the initial rate (delta absorbance/min) by the slope of the standard curve (delta absorbance/µM) to get µM/min. This can also be written as µmoles/min/liter, which is also units/L.
What color is pNPP?
Is pNPP an enzyme?
p-Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases.
What is the clinical significance of alkaline phosphatase?
Background: Alkaline phosphatase (ALP) is an enzyme that is elevated by various hepatobiliary diseases. Generally its elevation is thought to indicate bile stasis. There are some reports that show ALP is an important prognostic factor for several cancers such as colon, lung, and gastric cancer.
What type of enzyme is alkaline phosphatase?
homodimeric protein enzyme
Alkaline phosphatase (ALP, ALKP, ALPase, Alk Phos) (EC 3.1. 3.1), or basic phosphatase, is a homodimeric protein enzyme of 86 kilodaltons. 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.
What is the advantage of pNPP assay?
The assay can be used for the quick analysis of the protein phosphatase activity under any non-standard conditions. The advantage of the PNPP phosphatase activity assay is that unlike radioactive assays the substrate concentration can be much higher than the K m.
How is pNPP phosphatase activity assayed?
The PNPP phosphatase activity is assayed in a reaction mixture (50 μl) containing 50 mM PNPP and a protein phosphatase buffer supplemented with additional components when required.
What is the typical km value for pNPP?
PNPP has apparent Km values for protein phosphatases in the range of 0.5 -10 mM (2-5). The PNPP phosphatase activity is assayed in a reaction mixture (50 μl) containing 50 mM PNPP and a protein phosphatase buffer supplemented with additional components when required.
What are the limitations of the radioactive PTP assay?
The radioactive PTP assay is the most sensitive one, and a not fully precipitated substrate or an accidental contact of the pipette with the non-hydrolyzed substrate pellet can lead to a high background. In order to calculate the specific activity of the PTP, the protein amount needs to be determined.