Application

This kit can be used for detection of ALP activity in serum (plasma), tissue, cells and other sample. 

Detection significance

Alkaline phosphatase (ALP) is a group of cytomembrane-related enzymes with hydrolysis and transfer activity, acting on a variety of phosphate substrates. ALP is a homologous dimerase and each catalytic site contains three metal ions. There are four isozymes in humans: tissue nonspecific ALP, intestinal ALP, placental ALP and genital cell ALP

Detection principle

Alkaline phosphatase decompose benzene disodium phosphate to produce free phenol and phosphoric acid. Phenol react with 4-aminopyrline in alkaline solution and oxidizes with potassium ferricyanide to form red quinone derivative. The enzyme activity can be calculated indirectly by measuring the OD value.

Experimental instrument

Test tube, Micropipettor, Vortex mixer, 37℃ water bath, Spectrophotometer (520 nm)

Sample preparation

1. Serum/plasma: Detect the sample directly. If the concentration is beyond the linear range, please dilute the sample with normal saline before detection.

2. Tissue: Accurately weigh the tissue sample, add 9 times the volume of PBS (0.01 M, pH7~7.4) according to the ratio of Weight (g): Volume (mL) =1:9. Mechanical homogenate the sample in ice water bath. Centrifuge at 10000 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

3. Culture cell sample: Wash the cells with PBS (0.01 M, pH7~7.4) for 1~2 times. Centrifuge at 1000 g for 10 min and then discard the supernatant and keep the cell sediment. Add PBS at a ratio of cell number (106): PBS (μL) =1: 300-500. Sonicate or grind with hand-operated in ice water bath. Centrifuge at 10000 g for 10 min, then take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1. Blank well: add 50 μL of double distilled water into a 5 mL EP tube.

Standard well: add 50 μL of 0.1 mg/mL Phenol standard application solution into a 5 mL EP tube.

Sample well: add 50 μL of Sample into a 5 mL EP tube.  

2. Add 500 μL of Reagent 1 and 500 μL of Reagent 2 respectively and mix with a vortex mixer.

3. Incubate at 37℃ for 15 min, then add 1500 μL of Reagent 3 immediately, mix fully.

4. Set spectrophotometer to zero with double distilled water and measure the OD values of each tube at 520 nm with 0.5 cm optical path cuvette.

Note: It can be refer to the following operating table

Sample (μL) 

Reagent 1 (μL) 

Reagent 2 (μL) 

Reagent 3 (μL) 

Technical parameters

1. The sensitivity of the kit is 0.2 King unit/100 mL.

2. The detection range of the kit is 0.2-55.6 King unit/100 mL.

3. The intra-assay CV is 2.1 % and the inter-assay CV is 5.6 %.

4. The recovery of the kit is 99 %.

Notes

1. This kit is for research use only.

2. Please progress strictly with operation procedures.

3. The validity of kit is 3 months.

4. Do not use components from different batches of kit.