Blood acetylcholinesterase activity colorimetric quantitative detection kit product specification (Chinese)
The main purpose
Blood 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, and light absorption occurs. The change in peak value is the authoritative and classical technical method of colorimetric determination of enzyme activity in blood samples. The technology has been carefully developed and successfully tested. It is suitable for the detection of acetylcholinesterase activity in various animal and human blood samples (plasma, serum, red blood cells, 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) 120 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 storage
15 ml conical centrifuge tube: container for sample preparation
(Micro) Benchtop Centrifuge: for sample preparation
Cuvette or 96-well plate: a container for colorimetry
Spectrophotometer or microplate reader: for colorimetric analysis
Experimental procedure
- Sample preparation
Option 1: plasma sample
- Prepare anticoagulant tubes such as heparin, ACD or EDTA
- Take 1 ml of blood and place it in an anticoagulation tube
- Place in a 4°C mini tabletop centrifuge for 10 minutes at a speed of 700g (or 3000RPM, eg eppendorf 5415)
- Carefully remove the upper yellow liquid into a new 1.5 ml centrifuge tube - this is the plasma component (note: avoid touching the white liquid layer )
- Immediately placed in an ice bath or placed in a -70 ° C freezer
- (Selecting step) Pipette 10 [mu] l quantitative protein assay (Note: We recommend using the Bradford Protein Assay Kit -30030.1 concentration)
Option 2: serum samples
1. Prepare a storage tube without anticoagulant
- Take 1 ml of blood and place it in the storage tube
- Allow to stand for 30 minutes at room temperature until blood clots
- Place in a 4°C mini tabletop centrifuge for 15 minutes at a speed of 2000g (or 5000RPM, eg eppendorf 5415)
- Carefully remove the upper yellow liquid into a new 1.5 ml centrifuge tube - this is the serum component (Note: Avoid touching the white liquid layer )
- Immediately placed in an ice bath or placed in a -70 ° C freezer
- (Selecting step) Pipette 10 [mu] l quantitative protein assay (Note: We recommend using the Bradford Protein Assay Kit -30030.1 concentration)
Option three : red blood cell sample
- Prepare anticoagulant tubes such as heparin, ACD or EDTA
- Take 1 ml of blood and place it in an anticoagulation tube
- Transfer to a 15 ml conical tube
- Place in a 4°C benchtop centrifuge for 30 minutes at a speed of 200g (or 1000RPM, eg eppendorf 5815)
- Carefully remove the supernatant
- Add 2 ml of cleaning solution (Reagent A) and mix
- Place in a 4°C benchtop centrifuge for 7 minutes at a speed of 1200g (or 2500RPM, eg eppendorf 5815)
- Carefully remove the supernatant
- Repeat the experiment steps 6 to 8 times
- Add 2 ml of pre-cooled lysate (Reagent B)
- Vortex oscillation for 15 seconds
- Place in a 4°C benchtop centrifuge for 7 minutes at a speed of 1200g (or 2500RPM, eg eppendorf 5815)
- Carefully remove the supernatant into a new 15 ml centrifuge tube
- Immediately placed in an ice bath or placed in a -70 ° C freezer
- (Selecting step) Pipette 10 [mu] l quantitative protein assay (Note: We recommend using the Bradford Protein Assay Kit -30030.1 concentration)
- 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
Microplate reader
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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