AChE activity in a quantitative colorimetric assay kit product specification (Chinese)
The main purpose
Tissue acetylcholinesterase activity colorimetric quantitative detection reagent is a product designed to release choline choline through an acetylcholinesterase reaction system, and after using Ellman's reagent, a yellow 5-mercapto-2-nitrobenzoic acid product is produced, which absorbs light. The change in peak value is the authoritative and classical technique for the determination of enzyme activity in cell lysing samples using colorimetry. The technology has been carefully developed and successfully tested. It is suitable for the detection of acetylcholinesterase activity in various tissue lysate suspension samples (animal, human, etc.). The product is strictly sterile, ready to use, simple in operation and stable in performance.
technical background
Acetylcholinesterase (AchE; EC 3.1.1.7), also known as erythrocyte cholinesterase, is widely found in eukaryotes, including certain plants, especially in brain tissue, neuromuscular junctions ( Neuromuscular junction), central nervous system cholinergic synapses and erythrocyte membranes are highly expressed, catalyzing the hydrolysis of the main neurotransmitter acetylcholine, producing acetic acid and choline, and terminating synaptic transmission. Its function is to regulate the action of acetylcholine. Neurotoxic gases, such as organophosphate (Sarin), inhibit acetylcholinesterase activity, cause neuromuscular spasm, and eventually asphyxiation. The serum acetylcholinesterase concentration is significantly reduced in association with organophosphorus pesticide poisoning, so blood testing is an important indicator of environmental pollution. Based on the synthetic substrate iodide thiothiocholine iodide, under the action of acetylcholinesterase, hydrolyzed to produce acetic acid and thiocholine, and Ellman reagent 5,5-dithio-bis(2-nitrate After the reaction of [5,5'-dithiobis-(2-nitrobenzoic acid); DTNB], a yellow 5-thio-2-nitrobenzoic acid (TNB) is produced. The activity of acetylcholinesterase was quantitatively analyzed by the change in its absorption peak (wavelength at 412 nm). The acetylcholinesterase reaction system is:
Acetylcholinesterase
Acetylthiocholine iodide → acetate + thiocholine
DTNB + thiocholine → TNB + choline-SS-TNB
product content
Cleaning solution (Reagent A) 60 ml
Lysate (Reagent B) 10 ml
Buffer (Reagent C) 40 ml
Reaction solution (Reagent D) 2 ml
Substrate solution (Reagent E) 4 ml
Negative solution (Reagent F) 2 ml
Product manual 1 copy
storage method
Store lysate (Reagent B), reaction solution (Reagent D) and substrate solution (Reagent E) in -20 ° C refrigerator; the rest are stored in 4 ° C refrigerator; reaction solution (Reagent D) and substrate solution (Reagent E) Avoid lighting; effective guarantee for June
User-supplied
1.5 ml centrifuge tube: container for sample preparation and storage
15 ml conical centrifuge tube: container for sample preparation
Mini Tabletop Centrifuge: for sample preparation
Cuvette or 96-well plate: a container for colorimetry
Spectrophotometer or microplate reader: for colorimetric analysis
Experimental procedure
- Sample preparation
- Surgical removal of animal tissue and weighing to determine 500 mg tissue weight
- (selection step) into a pre-cooled 15 ml conical tube
- (Selection step) Add 3 ml of cleaning solution (Reagent A) for 1 wash
- Move into a liquid nitrogen cryotube
- Immediately put in a liquid nitrogen tank overnight
- The next day, it was taken out from the liquid nitrogen tank, and the tissue was ground into a powder (immediately) with a grinding rod (note: do not freeze the tissue )
- Put in a 15 ml conical tube
- Add pre-cooled 500 μl lysate (Reagent B)
- Transfer to pre-cooled 1.5 ml centrifuge tube
- Strong vortex for 30 seconds, mix thoroughly
- Incubate in an ice bath for 30 minutes, during which the vortex is vortexed for 30 seconds every 10 minutes (Note: If you need to temporarily stop, put it in the -70 °C refrigerator for storage )
- Place in a 4°C mini tabletop centrifuge for 5 minutes at a speed of 16000g (or 13000RPM, eg eppendorf 5415)
- Carefully pipet 500 μl of supernatant into a new pre-cooled 1.5 mL tube
- Transfer 10 μl for protein quantification (Note: Bradford Protein Concentration Quantitation Kit -30030.1 is recommended )
- Immediately put in -70 ° C to save or place in the ice trough to continue the subsequent operations
- Preparation for measurement
- Turn on and set up the spectrophotometer (temperature is 37 ° C): wavelength 412nm, interval 5 minutes, reading 3 times (15 minutes total), and set zero
- Reagents removed from -20 ℃ refrigerator, an ice melting trough; reaction solution (Reagent D) and substrate solution (Reagent E) protected from light
- Buffer (Reagent C) equalizes temperature at room temperature; substrate solution (Reagent E ) is incubated at 50 ° C
- Sample background control
- Pipette 750 μl of buffer (Reagent C) into a new cuvette
- Add 100 μl of negative solution (Reagent F)
- Add 100 μl of substrate solution (Reagent E)
- Pour up and down several times and mix
- Incubate for 3 minutes at 37 ° C
- Add 50 μl of the sample to be tested (100 μg of protein) (Note: the sample should be clear )
- Pour up and down several times, mix (limited to 3 seconds)
- Immediately put into the spectrophotometer test, this is the sample background control (15 minutes reading - 0 minutes reading)
- Sample determination
- Pipette 750 μl of buffer (Reagent C) into a new cuvette
- Add 100 μl of reaction solution (Reagent D)
- Add 100 μl of substrate solution (Reagent E)
- Pour up and down several times and mix
- Incubate for 3 minutes at 37 ° C
- Add 50 μl of the sample to be tested (100 μg of protein) (Note: the sample should be clear )
- Pour up and down several times, mix (limited to 3 seconds)
- Immediately put into the spectrophotometer test, this is the sample reading (15 minutes reading - 0 minutes reading)
Fifth, calculate the sample activity
[(Sample reading - background reading) X 1 (system capacity; ml) X sample dilution factor] ÷ [0.05 (sample capacity; ml) X 13.6 (mmol absorbance) X 15 (reaction time; minutes)] = unit / ÷ ÷ (sample protein concentration) mg / ml = unit / mg
Unit = micromolar thiocholine / minute
- Then determined
- Mark the corresponding on the 96-well plate: sample background control and sample to be tested
- Transfer 190 μl of buffer (Reagent C ) to a 96-well plate.
- Add 25 μl of negative solution (Reagent F ) to the sample control well
- Add 25 μl of reaction solution (Reagent D ) to the sample well to be tested
- Add 25 μl of substrate solution (Reagent E ) to all wells
- Gently shake the 96-well plate
- Incubate for 3 minutes at 37 ° C
- Add 10 μl of the sample to be tested (20 μg protein) to all wells (note: the sample should be clear )
- Gently shake the 96-well plate
- Immediately put into the microplate reader test: 0 minute reading and 15 minute reading
[(Sample reading - background reading) X sample dilution factor X 0.25 (system capacity; ml)] ÷ [0.01 (sample capacity; ml) X 13.6 (mmol absorbance) X 0.6 (light path distance; cm) X 15 ( Reaction time; minutes)] = unit / ml ÷ (sample protein concentration) mg / ml = unit / mg
Unit = micromolar thiocholine / minute
Precautions
- This product is operated 20 times (cuvette) and 80 times (96-well plate)
- Wear gloves when handling
- Background determination for each sample
- It is recommended to use cuvette determination
- Samples must be clarified, it is vital
- Colorimetric determination within 3 seconds after loading
- The measured value changes from low to high; the measurement lasts for 15 minutes.
- After the colorimetric determination, the cuvette must be thoroughly cleaned.
- The sample is measured for 15 minutes and the reading is higher than 0 minutes.
- It is recommended that the sample protein concentration to be tested be 100 μg/50 μl (the company provides Bradford Protein Concentration Quantitation Kit -30030.1)
- If the concentration of the sample to be tested is too high or too low, the sample concentration can be adjusted.
- Acetylcholinesterase unit activity is defined as the amount of enzyme required to produce 1 micromole of thiocholine per minute at 37 ° C, pH 7.5 as an active unit
- The company provides a series of acetylcholinesterase detection reagent products
Quality Standard
- This product has been certified to be stable.
- This product has been identified and sensitive
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