Application

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

Detection significance

AchE is a serine hydrolase and widely exists in various animal tissues and serum. AchE can catalyze the hydrolysis of acetylcholine and play an important role in the regulation of nerve conduction.

Detection principle

AchE catalyzes the hydrolysis of acetylcholine to form choline, and choline react with dithio p-nitrobenzoic acid (DTNB) to form 5-mercapto-nitrobenzoic acid (TNB). TNB has an absorption peak at 412nm. And the activity of AchE is calculated by measuring the increasing rate of absorbance at 412nm.

Experimental instrument

Micro quartz cuvette/ Microplate (96 wells)

Centrifuge

Micropipettor

Vortex mixer

37℃ water bath

Spectrophotometer/Microplate reader (412 nm)

Extraction of crude enzyme solution

1. Preparation of bacteria, fungi or cells samples:

Collect the bacteria, fungi or cells samples into a centrifuge tube, then centrifuge the sample and discard the supernatant. Add Reagent 1 into the sediment according to the ratio of Bacteria fungi or cells number: Reagent 1 (mL) =500~1000: 1(it is recommended to add 1 mL of Reagent 1 into 5×106 cells), then treat the sample with sonication on ice (power: 300W, 3 seconds/time, interval for 7 seconds, the total process will be 3 min). Centrifuge the lysate at 8000g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection.

2. Preparation of tissue samples:

Add Reagent 1 into the tissue sample according to the ratio of Weight (g): Reagent 1 (mL) =1:5~10 (it is recommended to add 1 mL of Reagent 1 into 0.1 g tissue), then homogenize the sample on ice. Centrifuge the tissue homogenate at 8000g for 10 min at 4℃. Take the supernatant for detection.

3. Serum / plasma samples:

Detect the serum or plasma samples directly.

Detection procedures

1. Preheat the Spectrophotometer or Microplate Reader for 30 min, then adjust the wavelength at 412 nm and set to zero with distilled water.

2. Preheated reagent 2 in 37 ℃ water bath for 30 min.

3. Operation table

Note: Blank tubes should be measured only once.