Application

This kit can be used for detection of the lactase activity in tissue samples. This kit (50 Assays) can detect 24 samples.

Detection significance

Lactase is an enzyme produced by many organisms. It is located in the brush border of the small intestine of humans and other mammals. Lactase is essential to the complete digestion of whole milk; it breaks down lactose, a sugar which gives milk its sweetness. Lacking lactase, a person consuming dairy products may experience the symptoms of lactose intolerance. Lactase can be purchased as a food supplement, and is added to milk to produce "lactose-free" milk products. Lactase (also known as lactase-phlorizin hydrolase, or LPH), a part of the β-galactosidase family of enzymes, is a glycoside hydrolase involved in the hydrolysis of the disaccharide lactose into constituent galactose and glucose monomers. Lactase is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine. In humans, lactase is encoded by the LCT gene.

Detection principle

Lactase react with the corresponding substrate and produce monosaccharide, which produces hydrogen peroxide under the catalyzation of its oxidase. Hydrogen peroxide combines with the chromogenic agent to produce red products. Measure the OD value with spectrophotometer, and the lactase activity can be determined according to the measured OD value.

Experimental instrument

Tube, Micropipette, Vortex mixer, Centrifuge, Spectrophotometer (505 nm), 37℃ water bath

The preparation of tissue sample

10% tissue homogenate sample: 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 3500 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 table

1. Enzymatic reaction

2. Chromogenic reaction

1) Measured by microplate reader

Supernatant (μL)  

Chromogenic agent (μL) 

[Note] The optimum sampling concentration should be determined by pre-experiment before batch experiment, so that the (ODsample – ODcontrol) should be closed with ODstandard.

Note:

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

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

5. If the lactase activity is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).