Alkaline phosphatase electrophoresis

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. 

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). 

Sengelpv, H., Nielson, M. H., Borregaard, N. (1992). Separation of human neutrophil plasma membrane from vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis. J. Biol. Chem. 267,14912-17. 

Alkaliphilic Bacillus sp. AM-001 was aerobically grown to the early stationary phase at 37°C in alkaline medium (pH 9.0) containing 1% konjak mannan. Total chromosomal DNA obtained by the method of Saito and Miura( 14) was digested with Hindlll restriction enzyme. And 2 to 4 kbp DNA fraction of chromosomal DNA was collected by 1% agarose gel electrophoresis. The plasmid pUC19 was digested with Hindlll and then dephosphorylated with bacterial alkaline phosphatase. After the... 

Purified preparations of alkaline phosphatase from E. coli, judged homogeneous when examined in the analytical ultracentrifuge, contain several isozymes, because several bands which contain enzymic activity are obtained in starch-gel and disc-gel electrophoresis. Although most workers find three bands (38, 39, 41, 43, 69), four (44) and five (70) equally spaced bands have been found. 

Digest PCR product and vector according to standard protocols [21]. Dephos-phorylate the vector using alkaline phosphatase and purify the DNA by agarose gel electrophoresis. 

Figure 21. A. Reaction of Ca2+-ATPase protein to anti-PHB IgG. Purified Ca2+-ATPase protein (8.5 pg per lane) was separated by electrophoresis on a 10% polyacrylamide gel.30 Left lane 1 Coomassie blue stain of Ca2+-ATPase protein. Left lane 2 Western blot of Ca2+-ATPase protein probed with rabbit anti-PHB IgG. Second antibody was anti-rabbit IgG conjugated to alkaline phosphatase. Color development was with the alkaline phosphatase substrate kit (Bio-Rad). B. Phosphorylation of the Ca2+-ATPase by [32PjpolyP. Purified Ca2+-ATPase protein (2 jig) was phosphorylated at room temperature by [32P]polyP and separated by electrophoresis on a 10% polyacrylamide gel.30 Right lane 1 Coomassie blue stain of phosphorylated Ca2+-ATPase. Right lane 2 Autoradiogram of phosphorylated Ca2+-ATPase.

SINGLE ALKALINE PHOSPHATASE MOLECULE ASSAY BY CAPILLARY ELECTROPHORESIS LASER-INDUCED FLUORESCENCE DETECTION... 

In this paper we report the assaying of individual molecules of the enzyme alkaline phosphatase (EC 3.1.3.1) by capillary electrophoresis (CE) utilizing laser-... 

EN113 Lee, K.N., Costello, R., Kroll, M. and Elin, R.J. (1992). Comparison of heat inactivation and electrophoresis for evaluating isoenzymes of alkaline phosphatase. Clin. Chem. 38, 984, Abstr. 209. 

Figure 21-5 A, Poiyacrylam id e-gel electrophoresis of bone and liver alkaline phosphatases in human serum. Left, Mixture of two sera containing, respectively, entirely bone phosphatase and entirely liver phosphatase. Right, Mixture of the same two sera after each has been treated with neuraminidase for 0 minutes at 37 C.The anodai direction is downward.The more anodal zone is liver phosphatase. B, Densltometric scans of electrophoretic patterns shown in A. Broken line, Scan of mixture of untreated sera solid line, scan of mixture of sera treated briefly with neuraminidase. The anode is to the left. (From Moss DW, Edwords RK. In proved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion, with neuraminidose. Clin Chim Acta 1984 143 i 77-82.) ...

Mattiazzo M, Ramasamy I. Wheat germ lectin affinity electrophoresis of serum alkaline phosphatase with commercially available gels. Clin Chem 1993 39 1404-7. 

A biochemical evalution of human alkaline phosphatase is postponed until the above considerations have been presented. In our view, the most reasonable analytical approach is based on the measurement of L-phenyl-alanine-sensitive and -insensitive moieties along with their respective heat stabilities. To this may be added information gathered from starch-gel electrophoresis with native and heated serum and from the presence of L-phenylalanine-sensitive bands on the gels following electrophoresis. Experiments of a different type can be included, in which the serum is incubated with neuraminidase and susceptibility of the glycoprotein is established following electrophoresis. Finally, the data on L-phenyl-alanine inhibition of heat-sensitive and -insensitive moieties appear to make sense, if the population of normal subjects is divided into one with the slow-moving intestinal band and one without it. It is from this consideration and other indirect and direct inferences that the intestine is... 

Previous work on human alkaline phosphatases has utilized chromatography (ElO) and starch-gel electrophoresis. Thus in 1956 Boman and Westlund (B34) reported the purification and separation of serum phosphatases by Dowex-2 column chromatography. Moss (M34) used gel filtration on Sephadex G-200 and DEAE-celluIose chromatography for separating 5 -nucleotidase and nonspecific alkaline phosphatase activities in human sera. In most of the studies of alkaline phosphatases in human tissues of liver (M33), intestine (M34, M35), bone (M36), kidney (B46), and urine (B44, B46, B47), crude extracts of these tissues were used and... 

In this laboratory, attempts (G6, G8) have been made to purify and crystallize human placental alkaline phosphatase enzyme by a number of procedures involving homogenization with 0.05 M Tris buffer (pH 8.6), extraction with butanol, ammonium sulfate precipitation, exposure to heat, ammonium sulfate fractionation, dialysis, repeated ethanol fractionation, gel filtration with Sephadex G-200 (Fig. 18), continuous curtain electrophoresis on paper (Beckman Model CP), multiple TEAE-cellulose anion exchange chromatography, and equilibrium dialysis. Variant A (electrophoretically fast-moving) of human placental alkaline... 

We will now deal separately with the electrophoresis on paper, starch gel, agar gel, and Sephadex gel of alkaline phosphatase. 

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