Phosphatases substrates

Roy, A. V. Brown, M. E. and Hayden, J. E. Sodium thymolphthalein monophosphate, a new acid phosphatase substrate with greater specificity for the prostatic enzyme in serum. Clin. Chem. (1971), IJ, 1093-1102. 

M.S. Wilson and R.D. Rauh, Hydroquinone diphosphate an alkaline phosphatase substrate that does not produce electrode fouling in electrochemical immunoassays. Biosens. Bioelectron. 20, 276-283 (2004). 

The alkaline phosphatase substrates form precipitates based on either reduction of tetrazolium salts or on the production of colored diazo compounds. Substrates Vector Red, Vector Black, Vector Blue, and BCIP/NBT available from Vector Laboratories produce reaction products which are red, black, blue and purple/blue... 

Enzyme substrates- A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphosphatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl carbazole (AEC) are described. Other substrates can be found in textbooks of histochemistry (see also Chapter 24) a NBT/BCIP is made in advance, and stored at -20°C. After equilibration of 30 mL of alkaline phosphatase substrate buffer at 37°C, 10 mg of NBT are dissolved m 200 pL of dimethylformamide (DMF), and added to I mL of the prewarmed substrate buffer. The mixture is added dropwise to the remaining substrate buffer BCIP (5 mg), dissolved in 200 pL of DMF, is then added slowly, and the whole preparation stored m 4-mL aliquots at—20°C b AEC is prepared fresh daily by dissolving 2 mg of AEC in 1 2 mL of dimethyl-sulfoxide in a glass tube. The mixture is added to 10 mL of 20 mM acetate buffer, pH 5 0-5.2. Immediately prior to use, 1 pL of 30% (v/v) hydrogen peroxide is added The final mix may require filtration prior to use... 

Wash m alkaline phosphatase substrate buffer for 5 mm at room temperature. 

Alkaline phosphatase substrate buffer O.lMTris-HCl, 0 lMNaCl, 5 mMMgCl2,... 

Wash over 30 min as before, first with Tris/saline, and then with alkaline phosphatase substrate buffer. 

C. Fernandez-Sanchez and A. Costa-Garcia, 3-Indoxyl phosphate an alkaline phosphatase substrate for enzyme immunoassays with voltam-metric detection, Electroanalysis, 10 (1998) 249-255. 

One of the most sensitive method for detecting probe-target hybrids involves an alkaline phosphatase-conjugated anti-DIG antibody and chemoluminescent alkaline phosphatase substrates (Roche, Molecular Diagnostics). Following hybridiza-... 

Antibodies coated onto MTP wells capture kinase or phosphatase substrate phosphorylation state is detected by anti-phosphopeptide antibody coupled to detector dye can be read by time-resolved fluorescence (DELFIA) technique... 

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.

Cheung YW, Abell C, Balasubramanian S. A combinatorial approach to identifying protein tyrosine phosphatase substrates from a phosphotyrosine peptide library. I. Am. Chem. Soc. 1997 119 9568-9569. 

Phosphatase substrate />-nitrophenyl phosphate at 1 mg/mL in dd water. Prepare freshly as required. 

Add 200 pL/well of the phosphatase substrate solution to all 60 inner wells as in Step 9. Incubate the plate at room temperature in the dark for 30 min. 

Alkaline phosphatase substrate buffer 50 mMTris-HCl, 100 mMNaCl, 1 mMMgClj, pH 9.5. 

Enzyme substrates. A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphos-phatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl car-bazole (AEG) are described. Other substrates can be found in textbooks of histochemistry (see a o Chapter 10). 

Alkaline phosphatase substrate buffers For ELISA, 100 mM glycine, 1 mMMgClg and 1 mMZnCl2 pH 10.4 for Western blot, O.lMTris-HCl, O.lMNaCl, and 5 mMMgCl, pH 9.5. 

Min alkaline-phosphatase substrate, PBST wash, tap-water wash. 

Procedure for Serum, Intestinal, and Placental Alkaline Phosphatases—Substrate Phenyl Phosphate... 

Key words Phosphatase detection. Phosphate quantitation. Artificial and native phosphatase substrates. 

Fig. 3 Structures of chromogenic phosphatase substrates (a) Para-nitrophenyl phosphate (b) phenolphthalein monophosphate (c) thymolphthalein monophosphate...

Fig. 4 Structures of fluorogenic phosphatase substrates (a) 6,8-difluoro-4-methylumelliferyl phosphate (b) 3-0-methylfluorescein phosphate...

Fig. 5 Structures of luminogenic phosphatase substrates (a) CDP-star (b) o-luciferin 6 -0-phosphate...

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