Phosphatase assay

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

Data obtained from 16 replicate alkaline phosphatase assays that were measured within a single rotor Results are given in I.U., 30° C. 

Alkaline phosphatase assays based on 3-glycerophosphate now appears to be obsolete, and methods buffered by either glycine or barbital are also obsolete as these buffers inhibit ALP or are poor buffers. Serum alkaline phosphatase is known to be composed of several isoenzymes which presumably arise from bone, liver, intestine, and placenta. The placental alkaline phosphatase is known to be much more resistant to heat denaturation than the other isoenzymes, and this resistance provides a simple test for it (5). The other enzymes can be separated through the differential inhibition by phenylalanine, by electrophoresis and by specific antibodies. However, the clinical usefulness of the results obtained is in doubt (23). 

Wash the precipitates twice with the RIPA buffer, followed by one wash with the IX phosphatase assay buffer. 

Add 50(jlL of IX phosphatase assay buffer with the phosphotyrosine-labelled peptide. 

Figure 36.7. Example of Immusoft window appearing in the Analysis menu during the processing of the fluidic steps of an assay. The figure shows the electrochemical monitoring of the fluid flux within the microchannels obtained during the 30 loading steps of the microchannel coating procedure chosen for alkaline phosphatase assays.

A solution of substrate (50 pL/well) is added and the plates incubated in the absence of light (time varies from case to case, but is usually 15-30 min). After incubation, the reacted solution is collected in an ELISA plate by centrifuging at 335g for 1 min, and the absorbance of the samples read at the characteristic wavelength (e.g., 405 nm for alkaline phosphatase assays). 

Because of the complex and often overlapping principles behind kinase and phosphatase assays, I will review the principles of the various fluorescent and luminescent technologies. A textbook by Joseph R. Lakowitz (1999) titled Principles of Fluorescence Spectroscopy is recommended for detailed information on the biophysics of fluorescence. Olive (2004) and Von Ahsen and Boemer (2005) wrote good reviews on the advantages and disadvantages of various luminescent (including fluorescent) technologies for kinase assays. 

Kinase or phosphatase assays based on the AlphaScreen principle are similar to TR-FRET assays in that they usually require a biotinylated substrate peptide and an anti-phosphoserine or tyrosine antibody. These two reagents are sandwiched between biotin and protein A-functionalized acceptor and donor beads. A kinase assay would show an enzyme-dependent increase in antibody binding (and thus signal) over time and a phosphatase assay would show an enzyme-dependent decrease in antibody binding over time. In some cases, the phosphorylation of an epitope will block the antibody binding and thus a phosphatase assay in principle can be constructed as a signal increase assay (Von Leoprichting and Kumpf, 2004 Warner et al., 2004). 

Assay volumes usually range from 3 pL (for 1536-well MTPs) to 50 pL (384-well MTPs). Within a given total assay volume, smaller volumes of reagents are added. Frequently, we find it convenient to add reagents into the assay in equivalent volumes of assay buffer. As an example, for a 15-pL assay, one might add 5 pL of compound solution, 5 pL of enzyme stock solution, 5 pL of substrate mix, followed by 10 pL of quench solution in a stop buffer. For kinase assays, the stop buffer may be EDTA and for phosphatase assays, sodium orthovanadate. 

Parker, G.J. et al. 2000. Development of high-throughput screening assays using fluorescence polarization nuclear receptor-ligand-binding and kinase/phosphatase assays. J. Biomol. Screen. 5, 77-88. 

Rodems, S.M. et al. 2002. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev. Technol. 1, 9-19. 

Watanabe, T. et al. 1998. Synthesis of fluorescent substrates for protein tyrosine phosphatase assays. Bioorg. Med. Chem. Lett. 8, 1301-1302. 

The mouse bioassay, which is still the ofircial method used for some natural toxins present in the marine enviromnent, has been used as a routine screening method for a few years, but this method has been recently replaced in maty laboratories worldwide with a more sensitive method such as an enzyme-linked-immrmosorbent assay (Nagata 1995). Protein phosphatase assays (Metcalf 2001) have been also used but more details on these methods will be discussed later. 

The results obtained with a protein phosphatase assay and a HPLC/UV/MS method are compared with the results obtained with a bioluminescence assay, which is successfully introduced here for... 

Dyhrnian, S. T., and Palenik, B. (1999). Phosphate stress in cultures and field populations of the dinoflageUate Prorocentrum minimum detected by a single-ceU alkaline phosphatase assay. Appl. Environ. Microbiol. 65, 3205—3212. 

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