Alkaline Phosphatase Detection Kit | ||||
Product number | product name | package | ||
YJ0321 | Alkaline Phosphatase Detection Kit | 100 times |
Product introduction:
- Alkaline Phosphatase Assay Kit is a sample for the rapid and convenient detection of supernatants, serum, plasma, urine, etc. of lysed or homogenized products of cell or tissue samples. A kit for endogenous alkaline phosphatase activity.
- Alkaline Phosphatase (AP/ALP/AKP/ALKP/ALPase/Alk Phos), also known as alkaline phosphatase (EC 3.1.3.1), catalyzes the hydrolysis of phosphate linkages under basic conditions. In mammals, alkaline phosphatase activity in the liver, bile duct, kidney, bone and placenta is relatively high. Common alkaline phosphatase includes the intestine
Alkaline phosphatase (intestinal, ALPI), tissue-nonspecific isophosphatase (ALPL) and placental phosphatase (alkaline phosphatase, placental type, also known as placental alkaline) Phosphatase, PLAP). Common calf Intestinal Alkaline Phosphatase (CIAP/CIP) is widely used for labeling of secondary antibodies, etc., and is ultimately used for the detection of proteins and nucleic acids, and is also commonly used for DNA or RNA 5 Ì and 3 Ì ends. Dephosphorylation (demonophosphorylation), in particular the 5 Ì terminal dephosphorylation of the plasmid to avoid plasmid self-ligation.
- In stem cells, such as iPS, alkaline phosphatase is highly active and is often used as a marker for successful induction of iPS. In addition, the activity of alkaline phosphatase in differentiated colon cancer cells is also
Will be significantly elevated, as a qualitative and quantitative indicator of the degree of differentiation of colon cancer cells. In addition, elevated serum alkaline phosphatase, known as hyperalkaline phosphatasemia, is considered to be associated with malignant biliary obstruction, primary biliary cirrhosis, Hepatobiliary such as primary sclerosing cholangitis, hepatic lymphoma, and hepatic sarcoidosis
The disease is closely related. Elevated alkaline phosphatase activity in serum is also closely related to bone formation because alkaline phosphatase is a by-product of osteoblasts. Too low alkaline phosphatase activity in serum is also associated with some diseases. Alkaline phosphatase activity in serum of children and pregnant women is higher than that of ordinary people. Serum alkaline phosphatase activity ranges in
20-140U/L.
- Except for the alkaline phosphatase placental isoform, other endogenous alkaline phosphatase is easily inactivated after heating.
- The kit can detect alkaline phosphatase activity in supernatants, plasma, serum, urine or purified enzyme samples of lysed or homogenized products of cell or tissue samples.
- Para-nitrophenyl phosphate (pNPP) is a commonly used phosphatase chromogenic substrate that produces para-nitrophenol under alkaline conditions with alkaline phosphatase. Para-nitrophenol ( p -nitrophenol) is a yellow product under alkaline conditions and can be used to detect absorbance at 400-415 nm. The deeper the product, the higher the alkaline phosphatase assay activity, and vice versa. According to this, the level of alkaline phosphatase activity can be calculated by colorimetric analysis.
- Including standard and blank controls, this kit can be used to test 100 samples.
packing list:
Product number | product name | package | ||
P0321-1 | Detection buffer | 15ml | ||
P0321-2 | Chromogenic substrate | 2 tubes | ||
P0321-3 | P- nitrophenol solution (10 mM) | 0.1ml | ||
P0321-4 | Reaction stop solution | 12ml | ||
- | Instruction manual | 1 copy | ||
Storage Conditions:
Save at -20oC, valid for one year. The chromogenic substrate and the p- nitrophenol solution are stored in the dark.
Precautions:
- If you want to perform absolute quantification of enzyme activity, you must pay attention to accurate timing when performing enzymatic reactions. At this time, it is recommended to use a longer time such as incubation for 30 minutes to reduce the time error during the operation. At the same time, if the enzyme activity in the sample is high, the sample can be appropriately diluted in advance.
- Inhibitors of alkaline phosphatase such as EDTA, fluoride ion, and citrate should be avoided in the sample solution.
- The detection buffer and p- nitrophenol solution are harmful to the human body. Please be careful when handling, and pay attention to effective protection to avoid direct contact with the human body or inhalation. The reaction stop solution is corrosive. Please be careful when handling it, and pay attention to effective protection to avoid direct contact with the human body or other materials.
- This product is limited to scientific research by professionals, and should not be used for clinical diagnosis or treatment. It should not be used for food or medicine, and should not be stored in ordinary houses.
- For your safety and health, wear a lab coat and disposable gloves.
Instructions for use:
- Reagent preparation: Remove all reagents and return to room temperature for use.
- Chromogenic substrate solution: Take a tube of chromogenic substrate and dissolve in 2.5ml of detection buffer (can be dissolved first with 1ml detection buffer, fully dissolved and mixed, transferred to 15ml centrifuge tube, then added 1.5 Ml detection buffer), fully dissolved and mixed, placed on ice. Freshly prepared chromogenic substrate solution should be used within 6 hours.
b. Standard working solution: Take 10 μl of p- nitrophenol solution (10 mM), dilute to 0.2 ml with assay buffer, and the final concentration is 0.5 mM. | ||||||
2. | Sample preparation: | |||||
a. Preparation of cell or tissue lysate: lysis of cells or tissues with appropriate cell or tissue lysate, it is recommended to use a base of P0013J Western and IP cell lysis | ||||||
The liquid (without inhibitor) cleaves the relevant sample. If necessary, appropriate homogenization is required, followed by centrifugation to remove the supernatant for alkaline phosphatase detection. Note: in the lysate | ||||||
Can not contain phosphatase inhibitors. Samples can be frozen at -80oC, but repeated freeze-thaw cycles should be avoided. | ||||||
b. Preparation of plasma, serum and urine: Plasma and serum can be directly used in the determination of this kit after preparation according to conventional methods, but in order to eliminate the color of the sample itself. | ||||||
For interference, a control with plasma or serum but no substrate is required. Anticoagulant tubes containing EDTA and citrate should not be used in plasma preparation. Urine can usually also be straight | ||||||
Used for measurement. The above samples can be frozen at -80oC, but repeated freezing and thawing should be avoided. | ||||||
c. Dilution of the sample: If the sample contains a higher activity of alkaline phosphatase, it can be diluted with the original lysate or PBS, or in the kit. | ||||||
The assay buffer is diluted. If using the assay buffer provided in the kit for dilution, be careful to leave enough assay buffer for the kit. | ||||||
Measurement process. | ||||||
3. | Refer to the table below to set blank control wells, standard wells, and sample wells using a 96-well plate. Standards are used at 4, 8, 16, 24, 32 and 40 microliters, respectively. Samples are usually available. | |||||
Add 50 microliters directly. If the alkaline phosphatase activity in the sample is too high, the amount of the sample can be reduced or diluted as appropriate. | ||||||
Blank control (Blank) | Standard (Standard) | Sample (Sample) | ||||
Detection buffer | 50μl | (100-x) μl | (50-y) μl | |||
Chromogenic substrate | 50μl | - | 50μl | |||
sample | - | - | Yμl | |||
Standard working fluid | - | Xμl | - | |||
4. | Mix gently with a pipette tip or mix with a shaker. | |||||
5. | Incubate for 5-10 minutes at 37oC. (Note: When the alkaline phosphatase activity in the sample to be tested is low, the incubation time can be extended to 30 minutes) | |||||
6. | The reaction was stopped by adding 100 μl of a reaction stop solution to each well. At this point, the standard or wells with alkaline phosphatase activity will appear in different shades of yellow. | |||||
7. | The absorbance was measured at 405 nm. If 405 nm cannot be measured, the absorbance can also be detected in the range of 400-415 nm. If it cannot be measured immediately, it can be within a few hours. | |||||
Upon completion of the assay, the yellow color developed was stable within a few hours. | ||||||
8. | Alkaline phosphatase activity unit definition: Hydrolysis of para-nitrophenyl phosphate per minute at 37 ° C in diacetamine (DEA) buffer pH 9.8 | |||||
The amount of alkaline phosphatase required for the chromogenic substrate to produce 1 micromole of p- nitrophenol is defined as an enzyme activity unit, also referred to as a DEA enzyme activity unit. At pH 9.6 | ||||||
Alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate per minute to produce 1 micromole of p -nitrophenol in a glycine buffer at 25oC | ||||||
The amount is defined as an enzyme activity unit, also known as a Glycine enzyme activity unit. A Glycine enzyme activity unit is equivalent to approximately 3 DEA enzyme activity units. This test | ||||||
The kit measures the unit of DEA enzyme activity. | ||||||
9. | The alkaline phosphatase activity in the sample was calculated based on the enzyme activity definition. | |||||
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