Alkaline Phosphatase Activity Colorimetric Assay Kit | K412 (2023)


an offer ab83369
Cat # +Size K412-500
Maat 500 analyses
Detection method Absorption (405nm)
Kind of reactivity Mammal
applications The kit can detect 10-250 µU ALP in samples.
Features & Benefits • Simple process. takes ~3 hours
• Fast and convenient
• Analysis is sensitive and stable
• The TUNEL-based test kit contains complete components, including positive and negative control cells for easy detection of DNA fragmentation in cultured cells and tissue sections.
Kit parts • ALP Assay-buffer
• pNPP (10 TAB)
• ALP-enzym
• Stopoplossing
Storage conditions -20°C
Shipping conditions Gel packaging
USE For research purposes only! Not for human use.


Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in alkaline buffer and produces an organic radical and inorganic phosphate. Changes in the level and activity of alkaline phosphatase are associated with various diseases of the liver and bones. The BioVision Alkaline Phosphatase Assay Kit is a highly sensitive, simple, direct and HTS-ready colorimetric assay designed to measure ALP activity in serum and biological samples. Contains 10 substrate tablets for multiple use. The kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate that turns yellow (λmax= 405 nm) when dephosphorylated by ALP. The kit can detect 10-250 µU ALP in samples.

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We intend to use this kit with protein isolates that contain a protease inhibitor versus samples that do not contain a protease inhibitor. Are there any other ingredients that you should be aware of that could affect how this kit works if someone is taking protease inhibitors? Which protease inhibitor is recommended for use in this alkaline phosphatase assay kit?

The only chemicals to watch out for are EDTA, oxalate, fluoride, and citrate. There is no recommended protease inhibitor for this test kit. You can use cocktail K271-500.

After the pnPP solution is prepared, can I freeze the pnPP solution (e.g. at -20 degrees Celsius) for later use? If so, what is the maximum time I can store it at -20 degrees Celsius?

Unfortunately, the pNPP solution must be used within 12 hours of preparation. After collecting all samples, you can make the fresh pNPP solution and use it immediately.

We want to measure samples at different times. So in sample preparation, after adding ALP Assay Buffer to my cell samples, can I freeze those samples at -20 degrees Celsius?

Samples homogenized in assay buffer can be frozen in aliquots at -80°C until analysis

(Video) Alkaline Phosphatase (ALP) Activity Assay Kit Video | Biovision, Inc.

Can the incubation steps for pNPP and the ALP enzyme be combined into a single one-hour incubation step? Or it is necessary to have the two incubations take place separately.

Yes, you can incubate at the same time.

I was thinking about keeping the cell volume (and number) constant and varying the dose concentration of the drug I'm testing with this test. Why do you recommend different cell volumes?

We do not get a different volume of cells, but a different volume of the homogenized cell. So, in a nutshell, you spread an equal number of cells in each well, treat them with the drugs, then trypsinize the cells, extract the cell pellet, wash with ice-cold PBS, homogenize the resulting pellet in assay buffer, extract the supernatant, and use different volumes of this supernatant for subsequent analysis.

How do we normalize our latest measurements?

If you start with a variable number of cells, you can normalize against the total number of cells or the amount of protein used.

We are looking for a kit to detect secreted alkaline phosphatase reporter gene in serum samples. Can we use this kit?

To test for the gene, you must isolate DNA from the serum and use a PCR-based reaction to detect that specific gene. This assay is for the detection of enzyme activity. A DNA isolation kit of serum samples and some special SEAL primers would be ideal for you.

(Video) Colorimetric Analysis Of Alkaline Phosphatase Activity In S. aureus Biofilm l Protocol Preview

When the enzyme is resuspended and stored at 4 degrees Celsius, what is the reduction in its viability when used at times beyond the recommended two months, say three or four months later?

We have not checked the enzyme stability statistics at 4 degrees Celsius for over 2 months. Chances are it will remain stable for a bit longer, but I'm not exactly sure how long and how much of the stability/effectiveness is lost after that.

I would like to ask you what is the origin of the standard ALP enzyme that comes with the kit (human, bovine, recombinant)?

The enzyme comes from the intestine of the calf.

Can this kit be used with samples such as bacteria, plants, drosophila, yeast, etc.?

We optimized the kit using mammalian specimens. In theory, however, these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol must be adapted to the samples for effective results. Refer to this kit's references to see what species of specimens have been used with this kit in addition to mammalian specimens

Can we use frozen samples in this test?

Fresh samples are always preferable to frozen ones. However, frozen samples can also be used, provided they have been frozen immediately after isolation, have not been thawed multiple times (for which we recommend aliquoting the samples prior to freezing), and have been frozen for a relatively short period of time.

Can we use a different wavelength than the one proposed for the final analysis?

(Video) Western Blotting with Colorimetric Alkaline Phosphatase Detection

It is always recommended to use the exact recommended wavelength for the most effective results. However, most plate readers have flexibility in their detection bandwidth in +/- 10 nm steps. Depending on this flexibility range, you can deviate from the recommended wavelengths within certain limits.

What is the exact sample volume required for this test?

There is no specific volume we can recommend for the amount of sample to use as it is based entirely on the concentration and quality of the sample. You must perform a trial run with multiple sample volumes to determine the optimal volume that will give a reading within the linear range of the standard curve. Check out the reviews for this product to see what other customers have used with similar sample types.

What is the shelf life of this kit?

This kit is valid in an unopened form for 12 months from the date of shipment when stored under proper temperature and conditions. After opening and reconstitution, some components of this kit can be stored for 2 months at -20 degrees Celsius. Please refer to the data sheet for storage information and shelf life of each component.

Can we buy individual parts of this kit?

Yes, you can purchase all kit parts without the whole kit. Refer to Cat#s listed on data sheet to order.

Do I have to do a standard curve for every expt I do or is one curve/kit enough?

Yes, I would highly recommend doing the templates every time you do the expt. There is always the possibility that something was done differently that day and we don't want conditions to differ between standards and samples.

Anandika Dhaliwal, Engineering Lineage Potency and plasticity of stem cells using epigenetic molecules.Sci Rep, november 2018; 30389989.

Mohamed Aboul Ezz et al., (2017) The effect of cyclodextrin with cholesterol on post-thawing buffalo sperm quality in relation to sperm DNA damage and ultrastructure,Reproductive Biology, 2017, 17:42-50

Lee, Sung-Il et al. (2017) Evaluation of the shape, viability, stemness and osteogenic differentiation of spheroid cells formed by human gingival stem cells and osteoprogenitor cells,Exp Ther Med. 2017 juni;13(6):3467-3473.

Dalia, Ali et al. (2016) Epigenetic library screen identifies Abexinostat as a novel regulator of adipocyte and osteoblastic differentiation of human skeletal stem cells (mesenchymal stem cells).The stem cell Transl Med. 2016 Aug;5(8):1036-47.

Chen et al., PDGFB-based stem cell gene therapy increases bone strength in the mouse.PNAS, July 2015; 112: E3893 - E3900.

Click for more references of this producthere

(Video) Alkaline Phosphatase Test | ALP Test


What is the alkaline phosphatase colorimetric activity assay kit? ›

Alkaline Phosphatase Assay Kit (Colorimetric) ab83369 is a highly sensitive, simple, direct and HTS-ready colorimetric assay designed to measure alkaline phosphatase (ALP) activity in serum and other mammalian samples.

What is a normal range for alkaline phosphatase? ›

Normal Results

The normal range is 44 to 147 international units per liter (IU/L) or 0.73 to 2.45 microkatal per liter (µkat/L). Normal values may vary slightly from laboratory to laboratory.

How do you calculate alkaline phosphatase enzyme activity? ›

ALP activity (U/ml) = A/V/T

Where A is amount of pNP generated by samples (in μmol), V is volume of sample added in the assay well (in ml), T is reaction time (in minutes).

What is the alkaline phosphatase activity assay? ›

In the alkaline phosphatase assay protocol, ALP cleaves the phosphate group of the non-fluorescent 4-Methylumbelliferyl phosphate disodium salt (MUP) substrate, resulting in an increased fluorescent signal (Ex/Em = 360nm/440nm) when the substrate is dephosphorylated.

What is the purpose of alkaline phosphatase test? ›

An alkaline phosphatase test is often used to screen for or help diagnose diseases of the liver or bones. The test may also help diagnose or monitor other health conditions.

What is the positive result of acid phosphatase test? ›

The enzyme acid phosphatase hydrolyses the substrate to the corresponding phenol and phosphate ion. The phenol formed is simultaneously coupled with a suitable diazonium salt as a chromogen to give a characteristic colored dye stuff, which is a positive test for the presence of a seminal stain.

When should I worry about alkaline phosphatase? ›

Values above 130 U/L are usually considered to be high.

Its major function is to protect your intestinal tract against bacteria, aid in digestion, breakdown fats and some B vitamins, and promote bone formation [1]. High levels of ALP in the blood may indicate bone, liver, or bile duct disease.

Should alkaline phosphatase be high or low? ›

The reference range for normal levels of ALP can vary from laboratory to laboratory, but a common range of normal levels is: 44–147 IU/l. Other labs may consider the normal range to be 30–120 IU/l.

Does high alkaline phosphatase mean liver disease? ›

High alkaline phosphatase (ALP) levels may indicate that there's damage to your liver or that you have a type of bone disorder. Liver damage creates a different type of ALP than bone disorders do.

What cancers are associated with high alkaline phosphatase? ›

Background: Alkaline phosphatase (ALP) is an enzyme that is elevated by various hepatobiliary diseases. Generally its elevation is thought to indicate bile stasis. There are some reports that show ALP is an important prognostic factor for several cancers such as colon, lung, and gastric cancer.

What infections cause high alkaline phosphatase? ›

Extremely high elevations of alkaline phosphatase are most frequently seen in patients with sepsis, malignant obstruction, and AIDS. Patients with sepsis can have an extremely high alkaline phosphatase level and a normal bilirubin.

What is Wilson's liver disease? ›

Wilson disease is a genetic disorder that prevents the body from removing extra copper, causing copper to build up in the liver, brain, eyes, and other organs. Without treatment, high copper levels can cause life-threatening organ damage.

What does phosphatase activity mean? ›

Phosphatase activity refers to the actions of two complementary, but distinct enzymes: phosphodiesterase (PDE) and phosphomonoesterase (PME). PDE hydrolyses complex organic P compounds such as nucleic acids and phospholipids into phosphomonoesters (mononucleotides and inositol phosphates).

What is alkaline phosphatase in inflammation? ›

Alkaline phosphatase (AP) is an enzyme that exhibits anti-inflammatory effects by dephosphorylating inflammation triggering moieties (ITMs) like bacterial lipopolysaccharides and extracellular nucleotides.

Which disease is diagnosed by assaying acid phosphatase activity? ›

Abstract. Serum acid phosphatase activity is the most commonly measured biochemical marker for diagnosing prostatic cancer and monitoring responses of such patients to therapy.

What does it mean if alkaline phosphatase is low? ›

Low levels of ALP indicate a deficiency in zinc and magnesium but can also be an indicator of a rare genetic disease called hypophosphatasia (HPP), which affects bones and teeth.

Can fatty liver cause high alkaline phosphatase? ›

Further, although the alkaline phosphatase can be elevated in fatty liver (yours is just a bit high), it is more common for AST and ALT to be elevated, which yours are not. An ultrasound scan is a good, but not definitive, way of looking for fatty liver.

What are the symptoms of low alkaline phosphatase? ›

Symptoms of Low Alkaline Phosphatase
  • Headache, breathlessness, and fatigue (if you have B12 deficiency and anemia) [2]
  • Weight gain, muscle weakness, and dry skin (if you have hypothyroidism) [3]
Dec 15, 2022

What disease is related to acid phosphatase? ›

Conditions associated with elevations in prostatic acid phosphatase (PAP) include the following:
  • Prostate cancer.
  • Benign prostatic hyperplasia.
  • Prostatic infarction.
  • Manipulation of the prostate through a massage or rectal examination.
May 18, 2023

What does acid phosphatase do in the liver? ›

Acid phosphatase is a ubiquitous lysosomal enzyme that hydrolyses organic phosphates at an acid pH. Although the postpuberteral prostatic epithelial cell contains a uniquely high concentration of acid phosphatase, cellular components of bone, spleen, kidney, liver, intestine, and blood also contain this enzyme.

What is acid phosphatase a tumor marker of? ›

Prostatic acid phosphatase (PAP) originates in the prostate and is normally present in small amounts in the blood. In addition to prostate cancer, elevated levels of PAP may indicate testicular cancer, leukemia, and non-Hodgkin's lymphoma, as well as some noncancerous conditions.

What liver problems cause high alkaline phosphatase? ›

A high alk phos level occurs when there is a blockage of flow in the biliary tract or a buildup of pressure in the liver--often caused by a gallstone or scarring in the bile ducts.

What causes alkaline phosphatase levels to be high symptoms? ›

For instance, your blood can show higher levels of ALP if one of the bile ducts that drains your liver becomes blocked. Conditions such as liver cancer, cirrhosis, and hepatitis can also cause ALP levels to rise. Bone disorders like Paget disease and healing fractures are other things that may affect your ALP levels.

Is alkaline phosphatase a tumor marker? ›

Serum ALP was found to be a valuable tumor marker with high specificity in osteosarcoma.

Can vitamin D deficiency cause high alkaline phosphatase? ›

Elevated serum alkaline phosphatase (ALP) level is an essential marker for the diagnosis of vitamin D deficiency (1). Some cases of vitamin D deficiency are diagnosed accidentally on the basis of elevated ALP levels. Therefore, cases without high ALP may be excluded from a diagnosis of vitamin D deficiency.

Can liver disease cause low alkaline phosphatase? ›

A low serum alkaline phosphatase (ALP) level is an uncommon finding in patients with chronic liver disease (CLD).

Is alkaline phosphatase elevated in kidney failure? ›

Higher serum alkaline phosphatase (ALP) levels have been associated with excess mortality in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) and end-stage renal disease (ESRD).

Can drinking cause high alkaline phosphatase? ›

Twenty patients with longstanding alcoholism and biopsy-proven alcoholic liver disease presented with marked elevation of serum alkaline phosphatase (in excess of four times the upper limit of normal).

What cancers cause high liver enzymes? ›

High levels can indicate tissue damage, such as liver or kidney disease, injury to the heart or other muscles, pancreatitis, some infections and some cancers, including lymphoma and leukemia.

Can autoimmune disease cause high alkaline phosphatase? ›

Our data suggest that the increase in ALP and ALP-10 closely reflects the abnormal activation of T lymphocytes that is common in autoimmune diseases, and that the source of the ALP-10 is activated T lymphocytes.

Does high alkaline phosphatase mean leukemia? ›

Alkaline phosphatase enzyme is often elevated in the leukemic cells in chronic myelogenous leukemia. Abnormally high levels may also indicate disease in bone or liver, bile duct obstruction, or certain malignancies.

How do you lower alkaline phosphatase? ›

How to naturally reduce Alkaline Phosphatase?
  1. Support Your Liver. If your alkaline phosphatase levels are high, you could look into natural ways to support the health of your liver. ...
  2. Omega-3 Fatty Acids. ...
  3. Stop Smoking. ...
  4. Coffee. ...
  5. Resistance Exercise. ...
  6. Sun Exposure/Vitamin D.
Dec 2, 2022

How can you tell if someone has Wilson's disease? ›

Doctors typically use blood tests and a 24-hour urine collection test to diagnose Wilson disease. Doctors may also use a liver biopsy and imaging tests.

What can be mistaken for Wilson's disease? ›

Wilson disease is often confused with other liver diseases such as alcoholic liver disease, fatty liver disease due to obesity, or acute liver failure due to hepatitis viruses or drugs/toxins. It can also be confused with other neurological diseases, such as Parkinson's disease or multiple sclerosis.

What is the classic physical symptom of Wilson disease? ›

Fatigue, lack of appetite or abdominal pain. A yellowing of the skin and the whites of the eye (jaundice) Golden-brown eye discoloration (Kayser-Fleischer rings) Fluid buildup in the legs or abdomen.

Why is phosphatase activity important? ›

Phosphatase activity is one of several mechanistic responses to P deficiency in the plant-soil system, enabling the mineralization of organic P to increase P availability for both plants and soil organisms.

What increased the activity of alkaline phosphatase? ›

High alkaline phosphatase levels can occur if the bile ducts are obstructed. Also, the level of alkaline phosphatase increases if there is active bone formation occurring, as the enzyme is a byproduct of osteoblast activity (such as the case in Paget's disease of bone).

What is another name for alkaline phosphatase? ›

1–p34. It is expressed at its highest levels in liver, bone, and kidney (hence its alternate name L/B/K ALP) as well as at lower levels in various other tissues.

Does high alkaline phosphatase mean arthritis? ›

Increased concentration of serum alkaline phosphatase (ALP) is a common feature in rheumatoid arthritis (RA), although its origin remains unclear.

Can high alkaline phosphatase cause pain? ›

Second, elevated serum ALP could be associated with chronic pain.

What are two clinical disorders that cause the elevation of alkaline phosphatase activity in serum? ›

Bone pathology causes of elevated alkaline phosphatase include Paget's disease, hyperparathyroidism, osteomalacia, metastatic bone disease and a recent fracture.

What disease is indicated by a blood test returns elevated acid phosphatase levels? ›

Total acid phosphatase (AP) activity may be useful when evaluating for prostate cancer, Paget's disease, hyperparathyroidism with skeletal involvement, and Gaucher's disease since elevations in AP activity occur in these conditions.

What is ALP activity on hepatobiliary disease? ›

ALP is a leading biomarker of hepatobiliary injury in common preclinical species. Serum ALP levels increase when the patency of the bile duct is reduced, so ALP is widely used in nonclinical and human clinical settings as a marker of cholestatic liver injury.

What is colorimetric assay for enzyme activity? ›

Colorimetric Assays

This allows precise determination of the enzyme activity by optical density. For example, peroxidase activity is measured by the reduction of the substrate in the presence of hydrogen peroxide, producing a soluble chromogen that is measured with a spectrophotometer.

What is phosphatase test used for the analysis of? ›

Alkaline Phosphatase is an enzyme which is naturally present in milk, but is destroyed at a temperature just near to the pasteurization temperature. Alkaline Phosphatase test is used to indicate whether milk has been adequately pasteurised or whether it has been contaminated with raw milk after pasteurisation.

What is colorimetric enzyme assay? ›

Colorimetric assays use a colorimeter (spectrophotometer) to determine concentration of a chemical compound in a solution by measuring spectral absorbance of the compound at a particular wavelength. A color is formed during the reaction of a detection chemical with the target substance.

What is assay of acid phosphatase activity? ›

The assay is based on the hydrolysis of the p-nitrophenyl phosphate by intracellular acid phosphatases in viable cells to produce p-nitrophenol. For all cell types examined, absorbance of p-nitrophenol at 405 nm is directly proportional to the cell number in the range of 10(3)-10(5) cells.

What is colorimetric assay used to detect? ›

Colorimetric assays use reagents that undergo a measurable color change in the presence of the analyte. They are widely used in biochemistry to test for the presence of enzymes, specific compounds, antibodies, hormones and many more analytes.

What is a colorimetric test and what can you use it for? ›

Colorimetric analysis is a method of determining the concentration of a chemical element or chemical compound in a solution with the aid of a color reagent. It is applicable to both organic compounds and inorganic compounds and may be used with or without an enzymatic stage.

What is an example of a colorimetric assay? ›

The techniques reviewed include Coomassie Blue G-250 dye binding (the Bradford assay), the Lowry assay, the bicinchoninic acid assay and the biuret assay.

What is the most common cause of elevated alkaline phosphatase? ›

Causes of high ALP levels. ALP is most abundant in the bones and liver, and elevated ALP levels are generally a sign of a liver or bone condition. An obstruction of the liver or damage to it causes ALP levels to rise. Elevated levels can also result from an increase in bone cell activity.

What is the purpose of enzyme activity assay? ›

Enzymatic activity assays are predominately performed by researchers to identify the presence or quantity of a specific enzyme in an organism, tissue, or sample. Examples of such enzymes include α-amylase, catalase, laccase, peroxidase, lysozyme, and reporter enzymes alkaline phosphatase, and luciferase.

What are the different types of colorimetric assays? ›

Based on the chemistry involved, the two most common methods for the colorimetric detection and quantitation of total protein are the protein-dye binding assay and/or copper ion-based chelation assay.

What is enzyme assay for diagnosis? ›

Enzyme activity assays can be used for disease diagnosis and novel drug development validation. Understanding how well a key enzyme functions in a physiological process often reveals early signs of disease that has a long latency period.

What is the normal range for total acid phosphatase activity? ›

In adults and elderly persons, the normal findings for acid phosphatase are 0.13-0.63 U/L (Roy, Brower, Hayden; 37°C) or 2.2-10.5 U/L (SI units). Normal findings in children are 8.6-12.6 U/mL (30°C), while normal findings in newborns are 10.4-16.4 U/mL (30°C).


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