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Alkaline Phosphatase
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Alkaline phosphatases (APs) are highly ubiquitous enzymes, present in all species from bacteria to man. In humans, APs are encoded by a multi-gene family composed of four loci; i.e., tissue-nonspecific AP, also called bone/liver/kidney AP, (Weiss et al, J. Biol. Chem., 264, 12002-12010, 1988), intestinal (Henthorn et. al., J.Biol. Chem., 263, 12020-12027, 1988). The sequence and complexity of the AP genes from other vertebrates and lower species are now being elucidated. The biological function of AP isozymes is still unknown. In vitro, the enzymes behave as phosphotransferases at neutral pH. The use of phosphate acceptor molecules ( diethanolamine, tris, 2-amino-2-methyl-1-propanol) in the buffered substrate solutions increases the reaction rates and, thus, the sensitivity of assays based on AP determinations. The following scheme summarizes the general reaction catalyzed by APs:
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1.Source: |
Bovine Kidney
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
40 U/mg solid
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Protein (approx.): |
90% (biuret)
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Contaminants (approx.): |
Glutamate oxaloacetate transaminase <0.02%; gamma-glutamyl transferase <0.005%
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Catalog No.: |
057A0040
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2.Source: |
Bovine Kidney
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
750 U/mg solid
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Protein (approx.): |
90% (biuret)
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Contaminants (approx.): |
Glutamate oxaloacetate transaminase <0.02%; gamma-glutamyl transferase <0.005%
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Catalog No.: |
057A0750
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3.Source: |
Human Placenta
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
100 U/mg solid
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Protein (approx.): |
90% (biuret)
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Catalog No.: |
058A0100
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4.Source: |
Human Placenta
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
750 U/mg solid
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Protein (approx.): |
90% (biuret)
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Catalog No.: |
058A0750
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5.Source: |
Bovine Liver
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
100 U/mg protein
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Catalog No.: |
119A0100
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6.Source: |
Porcine Kidney
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
300 U/mg solid
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Protein (approx.): |
90% (biuret)
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Catalog No.: |
181A0300
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7.Source: |
Calf Intestine (Molecular Biology Grade)
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Form: |
50% Glycerol solution
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at 4° C (39° F); Do not freeze
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Activity (approx.): |
3,000-6,000 U/mg protein
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Contaminants (approx.): |
Free of endonuclease, exonuclease and RNase activities
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Catalog No.: |
235C4500
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8.Source: |
Calf Intestine (Molecular Biology Grade)
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Form: |
Ammonium sulfate suspension
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at 4° C (39° F); Do not freeze
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Activity (approx.): |
3,000-6,000 U/mg protein
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Contaminants (approx.): |
Free of endonuclease, exonuclease and RNase activities
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Catalog No.: |
235B4500
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9.Source: |
Calf Intestine (Conjugation Grade)
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Form: |
Ammonium sulfate suspension
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at 4° C (39° F); Do not freeze
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Activity (approx.): |
3,000-6,000 U/mg protein
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Catalog No.: |
140B4500
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10.Source: |
Calf Intestine (Conjugation Grade)
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Form: |
50% Glycerol solution
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at 4° C (39° F); Do not freeze
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Activity (approx.): |
3,000-6,000 U/mg protein
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Catalog No.: |
140C4500
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11.Source: |
Calf Intestine (Conjugation Grade)
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Form: |
3 M Sodium Chloride/TEA buffer
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at 4° C (39° F); Do not freeze
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Activity (approx.): |
3,000-6,000 U/mg protein
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Catalog No.: |
140E4500
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12.Source: |
Calf Intestine (Molecular Biology Grade)
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
3,000-6,000 U/mg protein
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Contaminants (approx.): |
Free of endonuclease, exonuclease and RNase activities
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Catalog No.: |
235A4500
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13.Source: |
Calf Intestine (Conjugation Grade)
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Form: |
Freeze-dried powder
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Solubility: |
Distilled water or dilute buffer
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Stability: |
Store at -20° C (-4° F)
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Activity (approx.): |
1,500-2,500 U/mg protein
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Catalog No.: |
140A4500
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The amount of enzyme which liberates one micromole of p-nitrophenol from p-nitrophenyl-phosphate per minute at 37°C, pH 9.8 in diethanolamine buffer.
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The increase in absorbance at Hg 405 nm is measured.
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- Diethanolamine buffer (1 mol/L; pH 9.8; MgCl2 0.5 mmol/L):
Dilute 10.6 grams (9.7 mLs) diethanolamine (99%) with distilled H2O, add 0.05 ml MgCl2 solution (2) and adjust the pH to 9.8 (at 37°C) with HCl, >= 2mol/L, adjust to 100 ml with distilled H2O. - Magnesium chloride solution (1 mol/L):
Dissolve 20.3g MgCl2●6 H2O in 100 ml distilled H2O - 4-Nitrophenyl phosphate solution (0.67 mol/L):
Weigh 250 mg 4-nitrophenyl phosphate, Na salt add 1.0 ml distilled H2O to dissolve. - Diluent (0.1 mol./L; pH 7.6):
Dissolve 1.86g TEA ●HCl in distilled H2O, add 0.1 ml MgCl2 solution (2) and 0.1 ml ZnCl2 (0.1 mol/L; prepare freshly); adjust the pH value to 7.6 with NaOH, 1 mol/L and adjust to 100 ml with distilled H2O. - Sample solution:
Dilute enzyme solution to an activity of 0.05 to 0.06 U/ml with buffer (4). Usual final dilutions will be in the 1:200,000 to 1:600,000 range. Let stand for approximately 15-20 min. at room temperature before conducting assay.
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- Set spectrophotometer(with temperature control) at 405 nm and 37°C.
- Into a cuvette pipette the following:
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Buffer(1) |
2.90 ml |
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4-nitrophenyl phosphate |
0.05 ml |
- Incubate cuvette in spectrophotometer at 37°C for 6-8 minutes to achieve temperature equilibration and establish blank rate, if any.
- Add 0.1 ml of diluted enzyme to the cuvette, mix and record increase in absorbance at 405 nm for 5 minutes.
- Calculate (ΔE405nm/min) from the linear portion of thE curve.
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| Activity (U/mg) = |
(ΔE405nm/min)(Total Vol.)(Enz. Diln.) |
| (18.5)(Enz. Vol.)(mg Enz./ml) |
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