CN104293884B - A kind of acetyl CoA contents measures test kit and method thereof - Google Patents
A kind of acetyl CoA contents measures test kit and method thereof Download PDFInfo
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- CN104293884B CN104293884B CN201410501212.8A CN201410501212A CN104293884B CN 104293884 B CN104293884 B CN 104293884B CN 201410501212 A CN201410501212 A CN 201410501212A CN 104293884 B CN104293884 B CN 104293884B
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Abstract
The invention discloses a kind of acetyl CoA contents and measure test kit and method thereof, this test kit includes: reagent one, is made up of Tris HCl, polyvinylpyrrolidone, mercaptoethanol, Phenylmethanesulfonyl fluoride and glycerol；Reagent two, is made up of malic dehydrogenase；Reagent three, is made up of citrate synthase；Reagent four, is made up of malic acid and oxidized form of nicotinamide-adenine dinucleotide；Reagent five, is made up of Tris HCl.Malic dehydrogenase can be catalyzed malic acid and oxidized form of nicotinamide-adenine dinucleotide generates oxaloacetic acid and DPNH, citrate synthase can be catalyzed S-acetyl-coenzyme-A and oxaloacetic acid generates citric acid and coenzyme A, utilize the coupling reaction of malic dehydrogenase and citrate synthase, the generating rate of acetyl CoA contents and prototype nadide is directly proportional, and under 340nm, the climbing speed of light absorption value has reacted the height of acetyl CoA contents.The present invention is easy and simple to handle, and detection sensitivity is high, and the response rate is high, significantly reduces reagent cost, present invention also simplifies detecting step, measures more simple and efficient.
Description
Technical field
The present invention relates to test kit field, be specifically related to a kind of acetyl CoA contents and measure examination
Agent box and method thereof.
Background technology
S-acetyl-coenzyme-A is widely present in animal, plant, microorganism and cultivation cell, is
A kind of important mesostate produced in organism energy substance metabolic process.Acetyl
It coenzyme A energy substance metabolism in vivo is the material of a pivotability.Sugar, fat, egg
White matter three major nutrient pools a common metabolic pathway-tricarboxylic by S-acetyl-coenzyme-A
Acid circulation and oxidative phosphorylation, generate carbon dioxide and water through this path exhaustive oxidation,
Release energy for the synthesis of ATP.Additionally, S-acetyl-coenzyme-A is synthetic fatty acid, ketoboidies,
The precursor substance of the biological active substances such as cholesterol and derivant thereof.Original S-acetyl-coenzyme-A
Detection method of content sensitivity is relatively low, it will usually because in sample, acetyl CoA contents is low, dry
Disturb many so that being difficult to detect.The most also do not have a kind of simple to operate, rapid sensitive at present
Test kit, it is possible to allow tester can measure the content of S-acetyl-coenzyme-A easily.
Summary of the invention
In order to meet the demand, it is desirable to provide a kind of acetyl CoA contents is surveyed
Determine test kit and method thereof, there is the features such as simple to operate, quick, special, sensitive, for
Tester provides the test kit of good quality and the operation sequence of optimization, through simple training
Instruction, tester just can be competent at this work.
For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the present invention is by following skill
Art scheme realizes:
A kind of acetyl CoA contents measures test kit, including following reagent:
Reagent one, in liquid, 4 DEG C of preservations, by polyvinylpyrrolidone (PVP), sulfydryl second
Alcohol, Phenylmethanesulfonyl fluoride (PMSF) and glycerol are dissolved in the Tris-HCl that concentration is 50mM, PH=7.5
Form after buffer solution, be placed in reagent bottle；
Reagent two, in powder ,-20 DEG C of preservations, it is made up of malic dehydrogenase (MDH),
It is placed in 1.5mLEP pipe；
Reagent three, in liquid, 4 DEG C of preservations, it is made up of citrate synthase, is placed in 1.5mLEP
Guan Zhong；
Reagent four, in powder ,-20 DEG C of preservations, by malic acid and oxidized form of nicotinamide-adenine dinucleotide (NAD)
Composition, is placed in reagent bottle；
Reagent five, in liquid, 4 DEG C of preservations, by the Tris-HCl that concentration is 50mM, PH=7.5
Buffer solution forms.
Preferably, in described reagent one, the quality of polyvinylpyrrolidone (PVP) is 2.5g,
The volume of mercaptoethanol is 37uL, and the concentration of Phenylmethanesulfonyl fluoride (PMSF) is 100mM, body
Amass as 500uL, the volume of glycerol be the concentration of 5mL, Tris-HCl buffer solution be 50mM,
PH=7.5, volume is 100mL；
In described reagent two, the quality of malic dehydrogenase (MDH) is 0.3mg；
In described reagent three, the volume of citrate synthase is 10uL；
In described reagent four, the quality of malic acid is 30mg and oxidized form of nicotinamide-adenine dinucleotide (NAD)
Quality is 7.5mg；
In described reagent five, the concentration of Tris-HCl buffer solution is 50mM, PH=7.5, body
Amass as 30mL.
A kind of acetyl CoA contents assay method using mentioned reagent box, including following step
Rapid:
Step 1. instrument and the preparation of articles for use；
Ultraviolet spectrophotometer or microplate reader, water-bath, desk centrifuge, adjustable shifting liquid
Device, trace quartz colorimetric utensil or 96 orifice plates, mortar, ice and distilled water；
The extraction of step 2. sample；
1) extract from antibacterial or cultivate cell: collect antibacterial or cultivate cell in centrifuge tube,
Abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 antibacterials or cultivation cell, use
Power is the ultrasound wave of 20%, to described antibacterial or the cultivation ultrasonic 3s of cell, is spaced 10s, weight
Multiple 30 times, broken described antibacterial or cultivation cell, then employing 13000g eccentricity, 4 DEG C
Centrifugal 10min, takes supernatant, puts the most to be measured；
2) extract from tissue: weigh about 0.1g tissue, add the described reagent one of 1mL,
Carry out ice bath homogenate, then use eccentricity 13000g, 4 DEG C of centrifugal 10min, take supernatant,
Put the most to be measured；
The preparation before use of step 3. reagent；
1) described reagent two adds reagent five described in 250uL, fully dissolve standby；
2) described reagent three adds reagent five described in 250uL, fully dissolve standby；
3) described reagent four adds reagent five described in 22.5mL, fully dissolve standby；
The preparation of step 4. working solution；
The working solution volume that calculating is intended, working solution volume=sample number × 0.23mL, according to
The working solution volume of described plan, by the described reagent two fully dissolved in above-mentioned steps 3,
Described reagent three and described reagent four mix according to the ratio of 1:1:90, or directly by above-mentioned step
The described reagent two and the described reagent three that have fully dissolved in rapid 3 join in described reagent four
Mixing；Before sample-adding, the described working solution configured is placed in water-bath preheating 10min.
Step 5. acetyl CoA contents measures；
1) spectrophotometer or microplate reader preheating 30min, return to zero at 340nm with distilled water；
2) the described working solution of 230uL and 25uL sample are taken to trace quartz colorimetric utensil or 96
Orifice plate, mixing；
3) light absorption value A2 during the light absorption value A1 and 80s of 20s at immediate record 340nm；
4) light absorption value Δ A=A2-A1 is calculated；
5) if using trace quartz colorimetric utensil to be measured, employing regression equation is y=1640x
+ 0.012, calculate acetyl CoA contents；If using 96 orifice plates to be measured, using and returning
Equation is y=3280x+0.024, calculates acetyl CoA contents；
Wherein, x represents that light absorption value Δ A, y represent standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working solution preheating is as follows:
1) if what described sample extracted from mammal, then sample-adding before, by configured
Described working solution is placed in 37 DEG C of water-baths preheating 10min；
2) if what described sample extracted from nonmammalian, then sample-adding before, by configuration well
Described working solution be placed in 25 DEG C of water-baths preheating 10min.
Preferably, in steps of 5, if using trace quartz colorimetric utensil to be measured, and press
Calculate method according to protein concentration, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/mg prot)=(1640 × Δ A+0.012) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Preferably, in steps of 5, if using trace quartz colorimetric utensil to be measured, and press
Quality calculates method in the same old way, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/mg prot)=(1640 × Δ A+0.012) ÷ (W
÷ V sample is total)=(1640 × Δ A+0.012) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Preferably, in steps of 5, if using trace quartz colorimetric utensil to be measured, and press
Calculate method according to antibacterial or cell density, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/104)=(1640 × Δ A+0.012) ÷ (400 ÷ V
Sample is total)=4.1 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
Preferably, in steps of 5, if using 96 orifice plates to be measured, and according to according to egg
White densitometer algorithm, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/mg prot)=(3280 × Δ A+0.024) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Preferably, in steps of 5, if using 96 orifice plates to be measured, and according to sample matter
Gauge algorithm, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/mg prot)=(3280 × Δ A+0.024) ÷ (W
÷ V sample is total)=(3280 × Δ A+0.024) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Preferably, in steps of 5, if using 96 orifice plates to be measured, and according to antibacterial or
Cell density calculates method, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/104)=(3280 × Δ A+0.024) ÷ (400 ÷
V sample is total)=8.2 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
The principle of the mensuration of the present invention is as follows:
Malic dehydrogenase (MDH) can be catalyzed malic acid and oxidized form of nicotinamide-adenine dinucleotide (NAD) generates
Oxaloacetic acid and DPNH (NADH).Citrate synthase can be catalyzed S-acetyl-coenzyme-A and
Oxaloacetic acid generates citric acid and coenzyme A.Utilize the idol of malic dehydrogenase and citrate synthase
Connection reaction, the generating rate of acetyl CoA contents and DPNH (NADH) is directly proportional,
Under 340nm, the climbing speed of light absorption value has reacted the height of acetyl CoA contents.
The invention has the beneficial effects as follows:
1, the detection method of the present invention utilizes the coupling of malic dehydrogenase and citrate synthase anti-
Should, the generating rate of acetyl CoA contents and NADH is directly proportional, by detecting the life of NADH
Become the height of speed indirect reaction acetyl CoA contents, detection sensitivity and original method phase
Ratio improves about 10 times, and lowest detection is limited to 1.6nmol/mL, solves because of second in sample
Acyl coenzyme A content is low, interference is many so that being difficult to the problem detected.
2, the test kit of the present invention is easy and simple to handle, uses spectrophotometer and microplate reader all can examine
Survey, shorten the response time.
3, the response rate of test kit of the present invention is up to 98%-102%, and mensuration system is
0.255mL, significantly reduces reagent cost.
4, this invention simplifies detecting step, simply preparing and adding by working solution before use
The specification that liquid is long-pending, measures more simple and efficient, and detection efficiency is greatly improved.
Described above is only the general introduction of technical solution of the present invention, in order to better understand this
The technological means of invention, and can be practiced according to the content of description, below with the present invention
Preferred embodiment describe in detail.The detailed description of the invention of the present invention is detailed by following example
Be given.
Detailed description of the invention
Below in conjunction with embodiment, describe the present invention in detail.
Embodiment 1
A kind of acetyl CoA contents measures test kit, including following reagent:
Reagent one, in liquid, 4 DEG C of preservations, are the polyvinylpyrrolidone of 2.5g by quality
(PVP), volume is the mercaptoethanol of 37uL, and concentration is 100mM, and volume is 500uL's
It is 50mM, PH=7.5 that Phenylmethanesulfonyl fluoride (PMSF) and the glycerol that volume is 5mL are dissolved in concentration,
Volume be 100mL Tris-HCl buffer solution after form, be placed in reagent bottle；
Reagent two, in powder ,-20 DEG C of preservations, are the malic dehydrogenase of 0.3mg by quality
(MDH) composition, is placed in 1.5mLEP pipe；
Reagent three, in liquid, 4 DEG C of preservations, it is made up of the citrate synthase that volume is 10uL,
It is placed in 1.5mLEP pipe；
Reagent four, in powder ,-20 DEG C of preservations, malic acid and quality by quality is 30mg are
Oxidized form of nicotinamide-adenine dinucleotide (NAD) composition of 7.5mg, is placed in 30mL reagent bottle；
Reagent five, in liquid, 4 DEG C of preservations, are 50mM by concentration, PH=7.5, and volume is
The Tris-HCl buffer solution composition of 30mL.
A kind of acetyl CoA contents assay method using mentioned reagent box, including following step
Rapid:
Step 1. instrument and the preparation of articles for use；
Ultraviolet spectrophotometer, water-bath, desk centrifuge, adjustable pipette, trace
Quartz colorimetric utensil, mortar, ice and distilled water；
The extraction of step 2. sample；
1) extract from antibacterial or cultivate cell: collect antibacterial or cultivate cell in centrifuge tube,
Abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 antibacterials or cultivation cell, use
Power is the ultrasound wave of 20%, to described antibacterial or the cultivation ultrasonic 3s of cell, is spaced 10s, weight
Multiple 30 times, broken described antibacterial or cultivation cell, then employing 13000g eccentricity, 4 DEG C
Centrifugal 10min, takes supernatant, puts the most to be measured；
2) extract from tissue: weigh about 0.1g tissue, add the described reagent one of 1mL,
Carry out ice bath homogenate, then use eccentricity 13000g, 4 DEG C of centrifugal 10min, take supernatant,
Put the most to be measured；
The preparation before use of step 3. reagent；
1) described reagent two adds reagent five described in 250uL, fully dissolve standby；
2) described reagent three adds reagent five described in 250uL, fully dissolve standby；
3) described reagent four adds reagent five described in 22.5mL, fully dissolve standby；
The preparation of step 4. working solution；
The working solution volume that calculating is intended, working solution volume=sample number × 0.23mL, according to
The working solution volume of described plan, by the described reagent two fully dissolved in above-mentioned steps 3,
Described reagent three and described reagent four mix according to the ratio of 1:1:90, or directly by above-mentioned step
The described reagent two and the described reagent three that have fully dissolved in rapid 3 join in described reagent four
Mixing；Before sample-adding, the described working solution configured is placed in water-bath preheating 10min.
Step 5. acetyl CoA contents measures；
1) spectrophotometer preheating 30min, returns to zero at 340nm with distilled water；
2) the described working solution of 230uL and 25uL sample are taken to trace quartz colorimetric utensil, mixing；
3) light absorption value A2 during the light absorption value A1 and 80s of 20s at immediate record 340nm；
4) light absorption value Δ A=A2-A1 is calculated；
5) using regression equation is y=1640x+0.012, calculates acetyl CoA contents；
Wherein, x represents that light absorption value Δ A, y represent standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working solution preheating is as follows:
1) if what described sample extracted from mammal, then sample-adding before, by configured
Described working solution is placed in 37 DEG C of water-baths preheating 10min；
2) if what described sample extracted from nonmammalian, then sample-adding before, by configuration well
Described working solution be placed in 25 DEG C of water-baths preheating 10min.
Preferably, in steps of 5, if calculating method according to protein concentration, then acetylcoenzyme is measured
The computing formula of A content is as follows:
Acetyl CoA contents (nmol/mg prot)=(1640 × Δ A+0.012) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Preferably, in steps of 5, if calculating method according to sample quality, then acetylcoenzyme is measured
The computing formula of A content is as follows:
Acetyl CoA contents (nmol/mg prot)=(1640 × Δ A+0.012) ÷ (W
÷ V sample is total)=(1640 × Δ A+0.012) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Preferably, in steps of 5, if calculating method according to antibacterial or cell density, then measure
The computing formula of acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/104)=(1640 × Δ A+0.012) ÷ (400 ÷ V
Sample is total)=4.1 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
Embodiment 2
A kind of acetyl CoA contents measures test kit, including following reagent:
Reagent one, in liquid, 4 DEG C of preservations, are the polyvinylpyrrolidone of 2.5g by quality
(PVP), volume is the mercaptoethanol of 37uL, and concentration is 100mM, and volume is 500uL's
It is 50mM, PH=7.5 that Phenylmethanesulfonyl fluoride (PMSF) and the glycerol that volume is 5mL are dissolved in concentration,
Volume be 100mL Tris-HCl buffer solution after form, be placed in reagent bottle；
Reagent two, in powder ,-20 DEG C of preservations, are the malic dehydrogenase of 0.3mg by quality
(MDH) composition, is placed in 1.5mLEP pipe；
Reagent three, in liquid, 4 DEG C of preservations, it is made up of the citrate synthase that volume is 10uL,
It is placed in 1.5mLEP pipe；
Reagent four, in powder ,-20 DEG C of preservations, malic acid and quality by quality is 30mg are
Oxidized form of nicotinamide-adenine dinucleotide (NAD) composition of 7.5mg, is placed in 30mL reagent bottle；
Reagent five, in liquid, 4 DEG C of preservations, are 50mM by concentration, PH=7.5, and volume is
The Tris-HCl buffer solution composition of 30mL.
A kind of acetyl CoA contents assay method using mentioned reagent box, including following step
Rapid:
Step 1. instrument and the preparation of articles for use；
Microplate reader, water-bath, desk centrifuge, adjustable pipette, 96 orifice plates, mortar,
Ice and distilled water；
The extraction of step 2. sample；
1) extract from antibacterial or cultivate cell: collect antibacterial or cultivate cell in centrifuge tube,
Abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 antibacterials or cultivation cell, use
Power is the ultrasound wave of 20%, to described antibacterial or the cultivation ultrasonic 3s of cell, is spaced 10s, weight
Multiple 30 times, broken described antibacterial or cultivation cell, then employing 13000g eccentricity, 4 DEG C
Centrifugal 10min, takes supernatant, puts the most to be measured；
2) extract from tissue: weigh about 0.1g tissue, add the described reagent one of 1mL,
Carry out ice bath homogenate, then use eccentricity 13000g, 4 DEG C of centrifugal 10min, take supernatant,
Put the most to be measured；
The preparation before use of step 3. reagent；
1) described reagent two adds reagent five described in 250uL, fully dissolve standby；
2) described reagent three adds reagent five described in 250uL, fully dissolve standby；
3) described reagent four adds reagent five described in 22.5mL, fully dissolve standby；
The preparation of step 4. working solution；
The working solution volume that calculating is intended, working solution volume=sample number × 0.23mL, according to
The working solution volume of described plan, by the described reagent two fully dissolved in above-mentioned steps 3,
Described reagent three and described reagent four mix according to the ratio of 1:1:90, or directly by above-mentioned step
The described reagent two and the described reagent three that have fully dissolved in rapid 3 join in described reagent four
Mixing；Before sample-adding, the described working solution configured is placed in water-bath preheating 10min.
Step 5. acetyl CoA contents measures；
1) microplate reader preheating 30min, returns to zero at 340nm with distilled water；
2) the described working solution of 230uL and 25uL sample are taken to 96 orifice plates, mixing；
3) light absorption value A2 during the light absorption value A1 and 80s of 20s at immediate record 340nm；
4) light absorption value Δ A=A2-A1 is calculated；
5) using regression equation is y=3280x+0.024, calculates acetyl CoA contents；
Wherein, x represents that light absorption value Δ A, y represent standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working solution preheating is as follows:
1) if what described sample extracted from mammal, then sample-adding before, by configured
Described working solution is placed in 37 DEG C of water-baths preheating 10min；
2) if what described sample extracted from nonmammalian, then sample-adding before, by configuration well
Described working solution be placed in 25 DEG C of water-baths preheating 10min.
Preferably, in steps of 5, if calculating method according to according to protein concentration, then second is measured
The computing formula of acyl coenzyme A content is as follows:
Acetyl CoA contents (nmol/mg prot)=(3280 × Δ A+0.024) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Preferably, in steps of 5, if calculating method according to sample quality, then acetylcoenzyme is measured
The computing formula of A content is as follows:
Acetyl CoA contents (nmol/mg prot)=(3280 × Δ A+0.024) ÷ (W
÷ V sample is total)=(3280 × Δ A+0.024) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Preferably, in steps of 5, if calculating method according to antibacterial or cell density, then measure
The computing formula of acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/104)=(3280 × Δ A+0.024) ÷ (400 ÷
V sample is total)=8.2 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
Above-described embodiment, simply to illustrate that the technology design of the present invention and feature, its objective is
It is to allow one of ordinary skilled in the art will appreciate that present disclosure real according to this
Execute, can not limit the scope of the invention with this.Every reality according to present invention
The change of the equivalence done by matter or modification, all should contain within the scope of the present invention.
Claims (10)
1. an acetyl CoA contents measures test kit, it is characterised in that include following examination
Agent:
Reagent one, in liquid, 4 DEG C of preservations, by polyvinylpyrrolidone, mercaptoethanol, benzene
After methanesulfonyl fluoride and glycerol are dissolved in the Tris-HCl buffer solution that concentration is 50mM, PH=7.5
Composition, is placed in reagent bottle；
Reagent two, in powder ,-20 DEG C of preservations, it is made up of malic dehydrogenase, is placed in 1.5mLEP
Guan Zhong；
Reagent three, in liquid, 4 DEG C of preservations, it is made up of citrate synthase, is placed in 1.5mLEP
Guan Zhong；
Reagent four, in powder ,-20 DEG C of preservations, it is made up of malic acid and oxidized form of nicotinamide-adenine dinucleotide,
It is placed in reagent bottle；
Reagent five, in liquid, 4 DEG C of preservations, by the Tris-HCl that concentration is 50mM, PH=7.5
Buffer solution forms.
Acetyl CoA contents the most according to claim 1 measures test kit, its feature
It is:
In described reagent one, the quality of polyvinylpyrrolidone is 2.5g, the body of mercaptoethanol
Amassing as 37uL, the concentration of Phenylmethanesulfonyl fluoride is 100mM, and volume is 500uL, the body of glycerol
Amassing the concentration for 5mL, Tris-HCl buffer solution is 50mM, PH=7.5, and volume is 100mL；
In described reagent two, the quality of malic dehydrogenase is 0.3mg；
In described reagent three, the volume of citrate synthase is 10uL；
In described reagent four, the quality of malic acid is that the quality of 30mg and oxidized form of nicotinamide-adenine dinucleotide is
7.5mg；
In described reagent five, the concentration of Tris-HCl buffer solution is 50mM, PH=7.5, body
Amass as 30mL.
3. the acetyl CoA contents using test kit as claimed in claim 2 measures
Method, it is characterised in that comprise the following steps:
Step 1. instrument and the preparation of articles for use；
Ultraviolet spectrophotometer or microplate reader, water-bath, desk centrifuge, adjustable shifting liquid
Device, trace quartz colorimetric utensil or 96 orifice plates, mortar, ice and distilled water；
The extraction of step 2. sample；
1) extract from antibacterial or cultivate cell: collect antibacterial or cultivate cell in centrifuge tube,
Abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 antibacterials or cultivation cell, use
Power is the ultrasound wave of 20%, to described antibacterial or the cultivation ultrasonic 3s of cell, is spaced 10s, weight
Multiple 30 times, broken described antibacterial or cultivation cell, then employing 13000g eccentricity, 4 DEG C
Centrifugal 10min, takes supernatant, puts the most to be measured；
2) extract from tissue: weigh 0.1g tissue, add the described reagent one of 1mL, enter
Row ice bath is homogenized, and then uses eccentricity 13000g, 4 DEG C of centrifugal 10min, takes supernatant, put
The most to be measured；
The preparation before use of step 3. reagent；
1) described reagent two adds reagent five described in 250uL, fully dissolve standby；
2) described reagent three adds reagent five described in 250uL, fully dissolve standby；
3) described reagent four adds reagent five described in 22.5mL, fully dissolve standby；
The preparation of step 4. working solution；
The working solution volume that calculating is intended, working solution volume=sample number × 0.23mL, according to
The working solution volume of described plan, by the described reagent two fully dissolved in above-mentioned steps 3,
Described reagent three and described reagent four mix according to the ratio of 1:1:90, or directly by above-mentioned step
The described reagent two and the described reagent three that have fully dissolved in rapid 3 join in described reagent four
Mixing；Before sample-adding, the described working solution configured is placed in water-bath preheating 10min；
Step 5. acetyl CoA contents measures；
1) spectrophotometer or microplate reader preheating 30min, return to zero at 340nm with distilled water；
2) the described working solution of 230uL and 25uL sample are taken to trace quartz colorimetric utensil or 96
Orifice plate, mixing；
3) light absorption value A2 during the light absorption value A1 and 80s of 20s at immediate record 340nm；
4) light absorption value Δ A=A2-A1 is calculated；
5) if using trace quartz colorimetric utensil to be measured, employing regression equation is y=1640x
+ 0.012, calculate acetyl CoA contents；If using 96 orifice plates to be measured, using and returning
Equation is y=3280x+0.024, calculates acetyl CoA contents；
Wherein, x represents that light absorption value Δ A, y represent standard concentration, and unit is nmo l/mL.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in step 4, the concrete grammar of described working solution preheating is as follows:
1) if what described sample extracted from mammal, then sample-adding before, by configured
Described working solution is placed in 37 DEG C of water-baths preheating 10min；
2) if what described sample extracted from nonmammalian, then sample-adding before, by configuration well
Described working solution be placed in 25 DEG C of water-baths preheating 10min.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using trace quartz colorimetric utensil to be measured and dense according to albumen
Degree calculating method, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/mg prot)=(1640 × Δ A+0.012) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using trace quartz colorimetric utensil to be measured, and according to sample matter
Gauge algorithm, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/mg prot)=(1640 × Δ A+0.012) ÷ (W
÷ V sample is total)=(1640 × Δ A+0.012) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using trace quartz colorimetric utensil to be measured, and according to antibacterial or
Cell density calculates method, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/104)=(1640 × Δ A+0.012) ÷ (400 ÷ V
Sample is total)=4.1 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using 96 orifice plates to be measured, and according to according to protein concentration meter
Algorithm, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/mg prot)=(3280 × Δ A+0.024) ÷ Cpr；
Wherein, Cpr represents sample protein concentration, and unit is mg/mL, needs additionally to measure.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using 96 orifice plates to be measured, and calculate method according to sample quality,
The computing formula then measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/mg prot)=(3280 × Δ A+0.024) ÷ (W
÷ V sample is total)=(3280 × Δ A+0.024) ÷ W；
Wherein, W represents sample quality, and unit is g；V sample always represents addition extracting liquid volume,
Volume is 1mL.
Acetyl CoA contents assay method the most according to claim 3, its feature exists
In, in steps of 5, if using 96 orifice plates to be measured, and according to antibacterial or cell density
Calculating method, then the computing formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmo l/104)=(3280 × Δ A+0.024) ÷ (400 ÷
V sample is total)=8.4 × Δ A；
Wherein, 400 represent antibacterial or cultivate total cellular score, and sum is 4,000,000.
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