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Alkaline Phosphatase (ALP)
AAT Bioquest offers a comprehensive portfolio of alkaline phosphatase (ALP) products including purified enzymes, fluorogenic and chromogenic substrates, and complete assay kits. Alkaline phosphatase is widely used as an enzyme reporter in ELISA and immunohistochemistry applications, and has also been employed in blotting workflows where stable, low-background detection is required. Our Amplite® assay kits and specialty substrates provide researchers with optimized solutions for detecting ALP activity across colorimetric, fluorimetric, and luminometric platforms.
Product Type | Detection Method | Best For | Key Products |
|---|---|---|---|
Complete Assay Kits | Colorimetric, Fluorimetric, Luminometric | Ready-to-use ALP activity assays, ELISA detection | Amplite® ALP Assay Kits |
Fluorogenic Substrates | Fluorescence (Blue, Green, NIR) | High-sensitivity detection, microplate assays | CF-MUP, DiFMUP, FDP, SunRed™ |
Chromogenic Substrates | Absorbance | ELISA, standard colorimetric detection | pNPP |
Chemiluminescent Substrates | Luminescence | Ultra-sensitive detection, Western blots | ReadiUse™ APS-5, D-Luciferin phosphate |
Enzymes & Conjugates | — | Dephosphorylation, biotin-based detection | CIAP, ALP-Streptavidin |
Amplite® Alkaline Phosphatase Assay Kits
The Amplite® ALP Assay Kits provide complete, optimized solutions for measuring alkaline phosphatase activity in microplate format. Each kit includes proprietary substrates, optimized buffers, and ALP standards, enabling sensitive and reproducible detection without extensive optimization.
- Multiple detection options — Choose colorimetric, fluorimetric (blue, green, or NIR), or luminometric readouts
- High sensitivity — Detect sub-picomolar ALP concentrations with optimized substrate chemistry
- Convenient format — All reagents included for immediate use in 96-well or 384-well plates
- Broad dynamic range — Linear response across wide concentration ranges
Fig. 1
Alkaline phosphatase dose response was measured with Amplite® Fluorimetric Alkaline Phosphatase Assay Kit in a solid black 96-well plate using a Gemini microplate reader (Molecular Devices). As low as 0.01 mU/well of alkaline phosphatase can be detected with 30 minutes incubation (n=3). RFU read at Ex/Em = 360/450 nm (Cutoff = 435 nm).
Fluorogenic ALP Substrates
Fluorogenic substrates offer superior sensitivity compared to colorimetric alternatives, making them ideal for detecting low levels of ALP activity. These substrates are non-fluorescent until cleaved by a phosphatase (i.e., ALP), producing a highly fluorescent product that can be quantified using a fluorescence microplate reader or fluorometer.
- CF-MUP — Superior alternative to standard MUP with better retention in living cells and enhanced signal stability
- DiFMUP — Fluorinated MUP analog with improved spectral properties and lower background
- FDP — Dual-phosphate substrate yielding bright green-fluorescent fluorescein with excellent spectral separation
- PhosLite™ Green — Generates photostable fluorescent precipitate for imaging phosphatase activity at cellular resolution
- SunRed™ Phosphate — Far-red/NIR emission (Ex 653/Em 661 nm) compatible with 633 nm laser excitation
Fig. 2
Detection of acid phosphatase activity CF-MUP and MUP. The concentration of the two substrates (initially approximately 10 uM) were matched by normalizing the obsorbance at 319 nm (pH = 10) to a value of 0.52 (assuming the extinction coefficient of each substrate was approximately equivalent). The resulting fluorescence signal was recorded using Ex/Em = 360/450 nm.
Chromogenic and Chemiluminescent Substrates
Chromogenic substrates like pNPP provide simple absorbance-based detection suitable for standard ELISA workflows. For applications requiring maximum sensitivity, chemiluminescent substrates such as ReadiUse™ APS-5 and D-Luciferin phosphate enable detection of sub-femtomole ALP levels.
- ReadiUse™ APS-5 — Ultrasensitive acridan-based chemiluminescent substrate detecting sub-femtomole ALP levels with extended signal duration
- D-Luciferin phosphate — Bioluminescent substrate for ALP detection via luciferase cascade, ideal for dual-reporter assays
- pNPP — Industry-standard chromogenic substrate for routine ELISA and colorimetric assays
ALP Enzymes and Related Reagents
In addition to ALP activity assay kits and substrates, AAT Bioquest also offers ALP enzymes, conjugates and reaction buffers. These include: calf intestinal alkaline phosphatase (CIAP), a workhorse enzyme for molecular biology applications including dephosphorylation of DNA/RNA ends to prevent self-ligation; ALP-streptavidin conjugates that enable sensitive detection in biotin-based assay systems; Signal Guard™ stopping solution which provides precise endpoint control for phosphatase assays.
This document (01.0078.211015r2) was last updated on Sat Feb 28 2026. All trademarks and registered trademarks mentioned herein are the property of their respective owners.