CN104293884A - Kit for and method for content determination of acetyl coenzyme A - Google Patents
Kit for and method for content determination of acetyl coenzyme A Download PDFInfo
- Publication number
- CN104293884A CN104293884A CN201410501212.8A CN201410501212A CN104293884A CN 104293884 A CN104293884 A CN 104293884A CN 201410501212 A CN201410501212 A CN 201410501212A CN 104293884 A CN104293884 A CN 104293884A
- Authority
- CN
- China
- Prior art keywords
- reagent
- acetyl coa
- sample
- coa contents
- volume
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit and a method for content determination of acetyl coenzyme A. The kit comprises a reagent I, a reagent II, a reagent III, a reagent IV and a reagent V, wherein the reagent I is prepared from Tris-HCl, polyvinylpyrrolidone, mercaptoethanol, phenylmethylsulfonyl fluoride and glycerinum; the reagent II is prepared from malic dehydrogenase; the reagent III is prepared from citrate synthase; the reagent IV is prepared from malic acid and an oxidation type coenzyme I; and the reagent V is prepared from Tris-HCl. The malic dehydrogenase can be used for catalyzing malic acid and the oxidation type coenzyme I to generate oxaloacetic acid and a reduced coenzyme I; the citrate synthase is used for catalyzing acetyl coenzyme A and oxaloacetic acid to generate citric acid and a coenzyme A; by virtue of a coupled reaction of the malic dehydrogenase and the citrate synthase, the content of the acetyl coenzyme A and the generation rate of the reduced coenzyme I are in a direct proportion; the climbing speed of the light absorption value at 340nm reflects the content of the acetyl coenzyme A. The kit disclosed by the invention is simple and convenient to operate, high in detection sensitivity and high in recovery rate, and the cost of the reagents is remarkably lowered. The detection steps are further simplified, so that the kit is simpler and more convenient and efficient to test.
Description
Technical field
The present invention relates to test kit field, be specifically related to a kind of acetyl CoA contents and measure test kit and method thereof.
Background technology
Acetyl-CoA is extensively present in animal, plant, microorganism and culturing cell, is a kind of important mesostate produced in organism energy substance metabolic process.Acetyl-CoA is the material of a pivotability in vivo in energy substance metabolism.Sugar, fat, protein three major nutrient pool a common metabolic pathway-tricarboxylic acid cycle and oxidative phosphorylation by acetyl-CoA, generate carbonic acid gas and water, release energy for the synthesis of ATP through this path exhaustive oxidation.In addition, acetyl-CoA is synthetic fatty acid, ketoboidies, the precursor substance of the physiologically active substance such as cholesterol and derivative thereof.Original acetyl CoA contents detection method sensitivity is lower, usually can because acetyl CoA contents in sample is low, disturb and be difficult to detect mainly with causing.Also there is no at present a kind of test kit of simple to operate, rapid sensitive on the market, tester can be allowed can to measure the content of acetyl-CoA easily.
Summary of the invention
In order to meet the demand, the present invention has aimed to provide a kind of acetyl CoA contents and has measured 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 schedule of operation of optimization, through simple training, tester just can be competent at this work.
For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of acetyl CoA contents measures test kit, comprises following reagent:
Reagent one, in liquid, 4 DEG C of preservations, being dissolved in concentration by polyvinylpyrrolidone (PVP), mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF) and glycerine is form after the Tris-HCl buffered soln of 50mM, PH=7.5, is placed in reagent bottle;
Reagent two, in pulvis ,-20 DEG C of preservations, are made up of malate dehydrogenase (malic acid dehydrogenase) (MDH), are placed in 1.5mLEP pipe;
Reagent three, in liquid, 4 DEG C of preservations, are made up of Oxalacetic transacetase, are placed in 1.5mLEP pipe;
Reagent four, in pulvis ,-20 DEG C of preservations, are made up of oxysuccinic acid and oxidized form of nicotinamide-adenine dinucleotide (NAD), are placed in reagent bottle;
Reagent five, in liquid, 4 DEG C of preservations, the Tris-HCl buffered soln being 50mM, PH=7.5 by concentration forms.
Preferably, in described reagent one, the quality of polyvinylpyrrolidone (PVP) is 2.5g, the volume of mercaptoethanol is 37uL, and the concentration of phenylmethylsulfonyl fluoride (PMSF) is 100mM, and volume is 500uL, the volume of glycerine is 5mL, the concentration of Tris-HCl buffered soln is 50mM, PH=7.5, and volume is 100mL;
In described reagent two, the quality of malate dehydrogenase (malic acid dehydrogenase) (MDH) is 0.3mg;
In described reagent three, the volume of Oxalacetic transacetase is 10uL;
In described reagent four, the quality of oxysuccinic acid is the quality of 30mg and oxidized form of nicotinamide-adenine dinucleotide (NAD) is 7.5mg;
In described reagent five, the concentration of Tris-HCl buffered soln is 50mM, PH=7.5, and volume is 30mL.
Adopt an acetyl CoA contents measuring method for mentioned reagent box, comprise the following steps:
The preparation of step 1. instrument and articles for use;
Ultraviolet spectrophotometer or microplate reader, water-bath, desk centrifuge, adjustable pipette, micro-quartz colorimetric utensil or 96 orifice plates, mortar, ice and distilled water;
The extraction of step 2. sample;
1) extract from bacterium or culturing cell: collection bacterium or culturing cell are in centrifuge tube, abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 bacteriums or culturing cell, adopt power to be the ultrasonic wave of 20%, to described bacterium or the ultrasonic 3s of culturing cell, interval 10s, repeats 30 times, broken described bacterium or culturing cell, then 13000g eccentricity is adopted, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
2) extract from tissue: take about 0.1g and organize, add the described reagent one of 1mL, carry out ice bath homogenate, then adopt eccentricity 13000g, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
The preparation before use of step 3. reagent;
1) add reagent two described in 250uL in described reagent two, fully dissolve for subsequent use;
2) add reagent two described in 250uL in described reagent three, fully dissolve for subsequent use;
3) add reagent two described in 22.5mL in described reagent four, fully dissolve for subsequent use;
The preparation of step 4. working fluid;
Calculate the working fluid volume intended, working fluid volume=sample number × 0.23mL, according to the working fluid volume of described plan, the described reagent two, described reagent three and the described reagent four that have fully dissolved in above-mentioned steps 3 are mixed according to the ratio of 1:1:90, or directly the described reagent two fully dissolved in above-mentioned steps 3 and described reagent three is joined mixing in described reagent four; Before application of sample, the described working fluid configured is placed in water-bath preheating 10 min.
Step 5. acetyl CoA contents measures;
1) spectrophotometer or microplate reader preheating 30min, return to zero in 340nm place with distilled water;
2) described working fluid and 25uL sample extremely micro-quartz colorimetric utensil or 96 orifice plates of 230uL are got, mixing;
3) the light absorption value A2 during light absorption value A1 of immediate record 340nm place 20s and 80s;
4) light absorption value Δ A=A2-A1 is calculated;
5) if use micro-quartz colorimetric utensil to measure, employing regression equation is y=1640x+0.012, calculates acetyl CoA contents; If use 96 orifice plates to measure, employing regression equation is y=3280x+0.024, calculates acetyl CoA contents;
Wherein, x represents light absorption value Δ A, and y represents standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working fluid preheating is as follows:
1) if described sample extracts from Mammals, then, before application of sample, the described working fluid configured is placed in 37 DEG C of water-bath preheating 10 min;
2) if described sample extracts from nonmammalian, then, before application of sample, the described working fluid configured is placed in 25 DEG C of water-bath preheating 10 min.
Preferably, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
Preferably, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
Preferably, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/10
4)=(1640 × Δ A+0.012) ÷ (400 ÷ V samples are total)=4.1 × Δ A;
Wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
Preferably, in steps of 5, if use 96 orifice plates to measure, and according to according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
Preferably, in steps of 5, if use 96 orifice plates to measure, and according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
Preferably, in steps of 5, if use 96 orifice plates to measure, and according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/10
4)=(3280 × Δ A+0.024) ÷ (400 ÷ V samples are total)=8.2 × Δ A;
Wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
The principle of mensuration of the present invention is as follows:
Malate dehydrogenase (malic acid dehydrogenase) (MDH) catalysis oxysuccinic acid and oxidized form of nicotinamide-adenine dinucleotide (NAD) can generate oxaloacetic acid and DPNH (NADH).Oxalacetic transacetase catalysis acetyl-CoA and oxaloacetic acid can generate citric acid and coenzyme A.Utilize the linked reaction of malate dehydrogenase (malic acid dehydrogenase) and Oxalacetic transacetase, the generating rate of acetyl CoA contents and DPNH (NADH) is directly proportional, and 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, detection method of the present invention utilizes the linked reaction of malate dehydrogenase (malic acid dehydrogenase) and Oxalacetic transacetase, the generating rate of acetyl CoA contents and NADH is directly proportional, by detecting the height of the generating rate indirect reaction acetyl CoA contents of NADH, detection sensitivity is compared with original method and is improve about 10 times, lowest detection is limited to 1.6nmol/mL, solving because acetyl CoA contents in sample is low, disturbing mainly with causing the problem being difficult to detect.
2, test kit of the present invention is easy and simple to handle, adopts spectrophotometer and microplate reader all can detect, shortens the reaction times.
3, the rate of recovery 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, by the simple and easy preparation of working fluid and the specification of liquid feeding volume before use, measure more simple and efficient, detection efficiency improves greatly.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, describe in detail below with preferred embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, describe the present invention in detail.
embodiment 1
A kind of acetyl CoA contents measures test kit, comprises following reagent:
Reagent one, in liquid, 4 DEG C of preservations, by the polyvinylpyrrolidone (PVP) that quality is 2.5g, volume is the mercaptoethanol of 37uL, and concentration is 100mM, it is 50mM that the glycerine of volume to be the phenylmethylsulfonyl fluoride (PMSF) of 500uL and volume be 5mL is dissolved in concentration, PH=7.5, volume is form after the Tris-HCl buffered soln of 100mL, is placed in reagent bottle;
Reagent two, in pulvis ,-20 DEG C of preservations, the malate dehydrogenase (malic acid dehydrogenase) (MDH) being 0.3mg by quality forms, and is placed in 1.5mLEP pipe;
Reagent three, in liquid, 4 DEG C of preservations, the Oxalacetic transacetase being 10uL by volume forms, and is placed in 1.5mLEP pipe;
Reagent four, in pulvis ,-20 DEG C of preservations, are made up of the oxidized form of nicotinamide-adenine dinucleotide (NAD) of quality to be the oxysuccinic acid of 30mg and quality be 7.5mg, are placed in 30mL reagent bottle;
Reagent five, in liquid, 4 DEG C of preservations are 50mM, PH=7.5 by concentration, and volume is the Tris-HCl buffered soln composition of 30mL.
Adopt an acetyl CoA contents measuring method for mentioned reagent box, comprise the following steps:
The preparation of step 1. instrument and articles for use;
Ultraviolet spectrophotometer, water-bath, desk centrifuge, adjustable pipette, micro-quartz colorimetric utensil, mortar, ice and distilled water;
The extraction of step 2. sample;
1) extract from bacterium or culturing cell: collection bacterium or culturing cell are in centrifuge tube, abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 bacteriums or culturing cell, adopt power to be the ultrasonic wave of 20%, to described bacterium or the ultrasonic 3s of culturing cell, interval 10s, repeats 30 times, broken described bacterium or culturing cell, then 13000g eccentricity is adopted, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
2) extract from tissue: take about 0.1g and organize, add the described reagent one of 1mL, carry out ice bath homogenate, then adopt eccentricity 13000g, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
The preparation before use of step 3. reagent;
1) add reagent two described in 250uL in described reagent two, fully dissolve for subsequent use;
2) add reagent two described in 250uL in described reagent three, fully dissolve for subsequent use;
3) add reagent two described in 22.5mL in described reagent four, fully dissolve for subsequent use;
The preparation of step 4. working fluid;
Calculate the working fluid volume intended, working fluid volume=sample number × 0.23mL, according to the working fluid volume of described plan, the described reagent two, described reagent three and the described reagent four that have fully dissolved in above-mentioned steps 3 are mixed according to the ratio of 1:1:90, or directly the described reagent two fully dissolved in above-mentioned steps 3 and described reagent three is joined mixing in described reagent four; Before application of sample, the described working fluid configured is placed in water-bath preheating 10 min.
Step 5. acetyl CoA contents measures;
1) spectrophotometer preheating 30min, returns to zero in 340nm place with distilled water;
2) described working fluid and the 25uL sample extremely micro-quartz colorimetric utensil of 230uL is got, mixing;
3) the light absorption value A2 during light absorption value A1 of immediate record 340nm place 20s and 80s;
4) light absorption value Δ A=A2-A1 is calculated;
5) adopt regression equation to be y=1640x+0.012, calculate acetyl CoA contents;
Wherein, x represents light absorption value Δ A, and y represents standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working fluid preheating is as follows:
1) if described sample extracts from Mammals, then, before application of sample, the described working fluid configured is placed in 37 DEG C of water-bath preheating 10 min;
2) if described sample extracts from nonmammalian, then, before application of sample, the described working fluid configured is placed in 25 DEG C of water-bath preheating 10 min.
Preferably, in steps of 5, if according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
Preferably, in steps of 5, if according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
Preferably, in steps of 5, if according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/10
4)=(1640 × Δ A+0.012) ÷ (400 ÷ V samples are total)=4.1 × Δ A;
Wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
embodiment 2
A kind of acetyl CoA contents measures test kit, comprises following reagent:
Reagent one, in liquid, 4 DEG C of preservations, by the polyvinylpyrrolidone (PVP) that quality is 2.5g, volume is the mercaptoethanol of 37uL, and concentration is 100mM, it is 50mM that the glycerine of volume to be the phenylmethylsulfonyl fluoride (PMSF) of 500uL and volume be 5mL is dissolved in concentration, PH=7.5, volume is form after the Tris-HCl buffered soln of 100mL, is placed in reagent bottle;
Reagent two, in pulvis ,-20 DEG C of preservations, the malate dehydrogenase (malic acid dehydrogenase) (MDH) being 0.3mg by quality forms, and is placed in 1.5mLEP pipe;
Reagent three, in liquid, 4 DEG C of preservations, the Oxalacetic transacetase being 10uL by volume forms, and is placed in 1.5mLEP pipe;
Reagent four, in pulvis ,-20 DEG C of preservations, are made up of the oxidized form of nicotinamide-adenine dinucleotide (NAD) of quality to be the oxysuccinic acid of 30mg and quality be 7.5mg, are placed in 30mL reagent bottle;
Reagent five, in liquid, 4 DEG C of preservations are 50mM, PH=7.5 by concentration, and volume is the Tris-HCl buffered soln composition of 30mL.
Adopt an acetyl CoA contents measuring method for mentioned reagent box, comprise the following steps:
The preparation of step 1. instrument and 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 bacterium or culturing cell: collection bacterium or culturing cell are in centrifuge tube, abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 bacteriums or culturing cell, adopt power to be the ultrasonic wave of 20%, to described bacterium or the ultrasonic 3s of culturing cell, interval 10s, repeats 30 times, broken described bacterium or culturing cell, then 13000g eccentricity is adopted, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
2) extract from tissue: take about 0.1g and organize, add the described reagent one of 1mL, carry out ice bath homogenate, then adopt eccentricity 13000g, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
The preparation before use of step 3. reagent;
1) add reagent two described in 250uL in described reagent two, fully dissolve for subsequent use;
2) add reagent two described in 250uL in described reagent three, fully dissolve for subsequent use;
3) add reagent two described in 22.5mL in described reagent four, fully dissolve for subsequent use;
The preparation of step 4. working fluid;
Calculate the working fluid volume intended, working fluid volume=sample number × 0.23mL, according to the working fluid volume of described plan, the described reagent two, described reagent three and the described reagent four that have fully dissolved in above-mentioned steps 3 are mixed according to the ratio of 1:1:90, or directly the described reagent two fully dissolved in above-mentioned steps 3 and described reagent three is joined mixing in described reagent four; Before application of sample, the described working fluid configured is placed in water-bath preheating 10 min.
Step 5. acetyl CoA contents measures;
1) microplate reader preheating 30min, returns to zero in 340nm place with distilled water;
2) described working fluid and 25uL sample to 96 orifice plate of 230uL is got, mixing;
3) the light absorption value A2 during light absorption value A1 of immediate record 340nm place 20s and 80s;
4) light absorption value Δ A=A2-A1 is calculated;
5) adopt regression equation to be y=3280x+0.024, calculate acetyl CoA contents;
Wherein, x represents light absorption value Δ A, and y represents standard concentration, and unit is nmol/mL.
Preferably, in step 4, the concrete grammar of described working fluid preheating is as follows:
1) if described sample extracts from Mammals, then, before application of sample, the described working fluid configured is placed in 37 DEG C of water-bath preheating 10 min;
2) if described sample extracts from nonmammalian, then, before application of sample, the described working fluid configured is placed in 25 DEG C of water-bath preheating 10 min.
Preferably, in steps of 5, if according to according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
Preferably, in steps of 5, if according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
Preferably, in steps of 5, if according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
Acetyl CoA contents (nmol/10
4)=(3280 × Δ A+0.024) ÷ (400 ÷ V samples are total)=8.2 × Δ A;
Wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
Above-described embodiment, just in order to technical conceive of the present invention and feature are described, its objective is and is one of ordinary skilled in the art can be understood content of the present invention and implement according to this, can not limit the scope of the invention with this.The change of every equivalence done by the essence of content of the present invention or modification, all should be encompassed in protection scope of the present invention.
Claims (10)
1.
?a kind of acetyl CoA contents measures test kit, it is characterized in that, comprises following reagent:
Reagent one, in liquid, 4 DEG C of preservations, being dissolved in concentration by polyvinylpyrrolidone, mercaptoethanol, phenylmethylsulfonyl fluoride and glycerine is form after the Tris-HCl buffered soln of 50mM, PH=7.5, is placed in reagent bottle;
Reagent two, in pulvis ,-20 DEG C of preservations, are made up of malate dehydrogenase (malic acid dehydrogenase), are placed in 1.5mLEP pipe;
Reagent three, in liquid, 4 DEG C of preservations, are made up of Oxalacetic transacetase, are placed in 1.5mLEP pipe;
Reagent four, in pulvis ,-20 DEG C of preservations, are made up of oxysuccinic acid and oxidized form of nicotinamide-adenine dinucleotide, are placed in reagent bottle;
Reagent five, in liquid, 4 DEG C of preservations, the Tris-HCl buffered soln being 50mM, PH=7.5 by concentration forms.
2. acetyl CoA contents according to claim 1 measures test kit, it is characterized in that:
In described reagent one, the quality of polyvinylpyrrolidone is 2.5g, and the volume of mercaptoethanol is 37uL, the concentration of phenylmethylsulfonyl fluoride is 100mM, and volume is 500uL, and the volume of glycerine is 5mL, the concentration of Tris-HCl buffered soln is 50mM, PH=7.5, and volume is 100mL;
In described reagent two, the quality of malate dehydrogenase (malic acid dehydrogenase) is 0.3mg;
In described reagent three, the volume of Oxalacetic transacetase is 10uL;
In described reagent four, the quality of oxysuccinic acid is the quality of 30mg and oxidized form of nicotinamide-adenine dinucleotide is 7.5mg;
In described reagent five, the concentration of Tris-HCl buffered soln is 50mM, PH=7.5, and volume is 30mL.
3. adopt an acetyl CoA contents measuring method for test kit as claimed in claim 2, it is characterized in that, comprise the following steps:
The preparation of step 1. instrument and articles for use;
Ultraviolet spectrophotometer or microplate reader, water-bath, desk centrifuge, adjustable pipette, micro-quartz colorimetric utensil or 96 orifice plates, mortar, ice and distilled water;
The extraction of step 2. sample;
1) extract from bacterium or culturing cell: collection bacterium or culturing cell are in centrifuge tube, abandon supernatant, add the described reagent one of 1mL according to every 4,000,000 bacteriums or culturing cell, adopt power to be the ultrasonic wave of 20%, to described bacterium or the ultrasonic 3s of culturing cell, interval 10s, repeats 30 times, broken described bacterium or culturing cell, then 13000g eccentricity is adopted, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
2) extract from tissue: take about 0.1g and organize, add the described reagent one of 1mL, carry out ice bath homogenate, then adopt eccentricity 13000g, 4 DEG C of centrifugal 10min, get supernatant, put to be measured on ice;
The preparation before use of step 3. reagent;
1) add reagent two described in 250uL in described reagent two, fully dissolve for subsequent use;
2) add reagent two described in 250uL in described reagent three, fully dissolve for subsequent use;
3) add reagent two described in 22.5mL in described reagent four, fully dissolve for subsequent use;
The preparation of step 4. working fluid;
Calculate the working fluid volume intended, working fluid volume=sample number × 0.23mL, according to the working fluid volume of described plan, the described reagent two, described reagent three and the described reagent four that have fully dissolved in above-mentioned steps 3 are mixed according to the ratio of 1:1:90, or directly the described reagent two fully dissolved in above-mentioned steps 3 and described reagent three is joined mixing in described reagent four; Before application of sample, the described working fluid configured is placed in water-bath preheating 10 min;
Step 5. acetyl CoA contents measures;
1) spectrophotometer or microplate reader preheating 30min, return to zero in 340nm place with distilled water;
2) described working fluid and 25uL sample extremely micro-quartz colorimetric utensil or 96 orifice plates of 230uL are got, mixing;
3) the light absorption value A2 during light absorption value A1 of immediate record 340nm place 20s and 80s;
4) light absorption value Δ A=A2-A1 is calculated;
5) if use micro-quartz colorimetric utensil to measure, employing regression equation is y=1640x+0.012, calculates acetyl CoA contents; If use 96 orifice plates to measure, employing regression equation is y=3280x+0.024, calculates acetyl CoA contents;
Wherein, x represents light absorption value Δ A, and y represents standard concentration, and unit is nmol/mL.
4.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in step 4, the concrete grammar of described working fluid preheating is as follows:
1) if described sample extracts from Mammals, then, before application of sample, the described working fluid configured is placed in 37 DEG C of water-bath preheating 10 min;
2) if described sample extracts from nonmammalian, then, before application of sample, the described working fluid configured is placed in 25 DEG C of water-bath preheating 10 min.
5.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
6.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
7.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use micro-quartz colorimetric utensil to measure, and according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
acetyl CoA contents (nmol/10
4
)=(1640 × Δ A+0.012) ÷ (400 ÷ V samples are total)=4.1 × Δ A;
wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
8.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use 96 orifice plates to measure, and according to according to protein concentration computing method, then the calculation 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 concn, and unit is mg/mL, needs to measure in addition.
9.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use 96 orifice plates to measure, and according to sample quality computing method, then the calculation 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 and adds extracting liquid volume, and volume is 1mL.
10.
acetyl CoA contents measuring method according to claim 3, is characterized in that, in steps of 5, if use 96 orifice plates to measure, and according to bacterium or cell density computing method, then the calculation formula measuring acetyl CoA contents is as follows:
acetyl CoA contents (nmol/10
4
)=(3280 × Δ A+0.024) ÷ (400 ÷ V samples are total)=8.4 × Δ A;
wherein, 400 represent bacterium or culturing cell sum, add up to 4,000,000.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410501212.8A CN104293884B (en) | 2014-09-26 | 2014-09-26 | A kind of acetyl CoA contents measures test kit and method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410501212.8A CN104293884B (en) | 2014-09-26 | 2014-09-26 | A kind of acetyl CoA contents measures test kit and method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104293884A true CN104293884A (en) | 2015-01-21 |
| CN104293884B CN104293884B (en) | 2016-08-17 |
Family
ID=52313832
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410501212.8A Active CN104293884B (en) | 2014-09-26 | 2014-09-26 | A kind of acetyl CoA contents measures test kit and method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104293884B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105651878A (en) * | 2015-12-31 | 2016-06-08 | 苏州科铭生物技术有限公司 | Kit and method for measuring content of ascorbic acid |
| CN108956500A (en) * | 2018-08-08 | 2018-12-07 | 大连大学 | A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method |
| CN109211817A (en) * | 2018-09-06 | 2019-01-15 | 苏州科铭生物技术有限公司 | A kind of CoA contents assay kit and its method based on micromethod |
| CN109324006A (en) * | 2018-11-12 | 2019-02-12 | 苏州科铭生物技术有限公司 | 1,3 diphospho glycerate acid content assay kit and its method based on micromethod |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101169442A (en) * | 2006-10-24 | 2008-04-30 | 苏州艾杰生物科技有限公司 | Carbon dioxide diagnosis/ determination reagent kit and carbon dioxide concentration determination method |
| CN102298040A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
| CN103743689A (en) * | 2014-01-03 | 2014-04-23 | 苏州科铭生物技术有限公司 | Kit for detecting content of coenzyme I NAD (Nicotinamide Adenine Dinucleotide) (H) |
-
2014
- 2014-09-26 CN CN201410501212.8A patent/CN104293884B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101169442A (en) * | 2006-10-24 | 2008-04-30 | 苏州艾杰生物科技有限公司 | Carbon dioxide diagnosis/ determination reagent kit and carbon dioxide concentration determination method |
| CN102298040A (en) * | 2010-06-25 | 2011-12-28 | 苏州艾杰生物科技有限公司 | Method for determining glycine and glycine determination kit |
| CN103743689A (en) * | 2014-01-03 | 2014-04-23 | 苏州科铭生物技术有限公司 | Kit for detecting content of coenzyme I NAD (Nicotinamide Adenine Dinucleotide) (H) |
Non-Patent Citations (2)
| Title |
|---|
| FLORA H.PETTIT: "Regulation of pyruvate dehydrogenase kinase and phosphatase by acetyl-CoA CoA and NADH NAD ratios", 《BIOCHEMICAL AND RESEARCH COMMUNICATIONS》, 31 December 1975 (1975-12-31) * |
| 曹春华等: "乙酰辅酶A在物质代谢中的作用", 《贵州师范大学学报(自然科学版)》, 2 April 1986 (1986-04-02) * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105651878A (en) * | 2015-12-31 | 2016-06-08 | 苏州科铭生物技术有限公司 | Kit and method for measuring content of ascorbic acid |
| CN108956500A (en) * | 2018-08-08 | 2018-12-07 | 大连大学 | A kind of low temperature malic dehydrogenase acetate concentration detection kit and its detection method |
| CN108956500B (en) * | 2018-08-08 | 2021-08-03 | 大连大学 | Low-temperature malate dehydrogenase acetic acid concentration detection kit and detection method thereof |
| CN109211817A (en) * | 2018-09-06 | 2019-01-15 | 苏州科铭生物技术有限公司 | A kind of CoA contents assay kit and its method based on micromethod |
| CN109324006A (en) * | 2018-11-12 | 2019-02-12 | 苏州科铭生物技术有限公司 | 1,3 diphospho glycerate acid content assay kit and its method based on micromethod |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104293884B (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104293884A (en) | Kit for and method for content determination of acetyl coenzyme A | |
| CN109971686A (en) | Caproic acid bacterium multiplication culture medium, caproic acid fermentation and caproic acid bacterium screening method | |
| CN107796770A (en) | A kind of isocitrate lyase Activity Assay Kit and its method | |
| CN103743689A (en) | Kit for detecting content of coenzyme I NAD (Nicotinamide Adenine Dinucleotide) (H) | |
| Tu et al. | iTRAQ quantitative proteomic analysis reveals lipid metabolism pathway of N+ ion-implanted C. pyrenoidosa cultivated in municipal wastewater | |
| CN108303475A (en) | A kind of detection method of energy matter | |
| CN102768190B (en) | Determining reagent of serum total bile acid and detecting method | |
| CN107782684A (en) | Codehydrogenase Ⅱ NADP+Or NADPH assays kit and its method | |
| CN107727644A (en) | A kind of (D) 2 hydroxyl glutaric acid detection kit | |
| CN107727589A (en) | A kind of Activity Assay Kit of transhydrogenase 1 and its method | |
| CN109913527A (en) | A method of utilizing shigella dysenteriae in ATP bioluminescence reaction detection food | |
| CN105506061A (en) | Kit and method for detecting activity of sucrose phosphate synthase | |
| CN109596550A (en) | A kind of content of trehalose assay kit and its method based on spectrophotometry | |
| CN102818801A (en) | Method for determining ATP | |
| CN104099397A (en) | Method for detecting cytotoxicity of nano materials through yeast mutant strains | |
| CN109211817A (en) | A kind of CoA contents assay kit and its method based on micromethod | |
| CN109470637B (en) | Method for determining activity of ethanol dehydrogenase | |
| CN101329328A (en) | Oxalic acid diagnosis / determination reagent kit and method for measuring oxalic acid concentration | |
| CN105567621B (en) | It is a kind of promote regenerating internal cell coenzyme NADP 11 genetic engineering cytoalgae and its application | |
| CN102532053B (en) | Preparation and purification methods of high-purity D-fluorescein | |
| CN101082572A (en) | Method for measuring aminoglutaric acid concentration and aminoglutaric acid diagnose reagent kit | |
| CN101082571A (en) | Method for measuring malic acid concentration and malic acid diagnose reagent kit | |
| CN105543331A (en) | Soil peroxidase activity test kit and method thereof | |
| CN202039067U (en) | Alimta medicine-targeted gene locus assay kit | |
| CN109632676A (en) | A method of the measurement intracapsular bacterium amount of sodium alginate micro gel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |