CN109324006A - 1,3 diphospho glycerate acid content assay kit and its method based on micromethod - Google Patents
1,3 diphospho glycerate acid content assay kit and its method based on micromethod Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000002253 acid Substances 0.000 title claims abstract description 16
- LJQLQCAXBUHEAZ-UWTATZPHSA-N 3-phospho-D-glyceroyl dihydrogen phosphate Chemical compound OP(=O)(O)OC[C@@H](O)C(=O)OP(O)(O)=O LJQLQCAXBUHEAZ-UWTATZPHSA-N 0.000 title claims abstract description 14
- 238000003149 assay kit Methods 0.000 title claims abstract description 11
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 34
- 238000001514 detection method Methods 0.000 claims abstract description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 18
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims abstract description 18
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims abstract description 18
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims abstract description 10
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 8
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 29
- XOHUEYCVLUUEJJ-UHFFFAOYSA-N 2,3-Bisphosphoglyceric acid Chemical compound OP(=O)(O)OC(C(=O)O)COP(O)(O)=O XOHUEYCVLUUEJJ-UHFFFAOYSA-N 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 16
- 235000018102 proteins Nutrition 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 239000012224 working solution Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- 239000010453 quartz Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- 230000008033 biological extinction Effects 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 230000031700 light absorption Effects 0.000 claims description 6
- 230000003287 optical effect Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 238000002525 ultrasonication Methods 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 241000124008 Mammalia Species 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 235000013311 vegetables Nutrition 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 claims description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 2
- 230000001351 cycling effect Effects 0.000 claims description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 2
- 230000003252 repetitive effect Effects 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 2
- 235000019441 ethanol Nutrition 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 3
- GHKCSRZBNZQHKW-UHFFFAOYSA-N 1-sulfanylethanol Chemical compound CC(O)S GHKCSRZBNZQHKW-UHFFFAOYSA-N 0.000 abstract description 2
- LMOIEPYPVQOZCT-UHFFFAOYSA-N 2,3-dihydroxypropanal;phosphoric acid Chemical class OP(O)(O)=O.OCC(O)C=O LMOIEPYPVQOZCT-UHFFFAOYSA-N 0.000 abstract description 2
- 150000007513 acids Chemical class 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 53
- 238000012856 packing Methods 0.000 description 2
- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 208000022329 Protein metabolism disease Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- XQRLCLUYWUNEEH-UHFFFAOYSA-N diphosphonic acid Chemical compound OP(=O)OP(O)=O XQRLCLUYWUNEEH-UHFFFAOYSA-N 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Physics & Mathematics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of 1,3- diphosphoglyceric acid assay kit and its method based on micromethod, the reagent of the kit includes the extracting solution that glycerol mixes, by MgSO by Tris-HCl, EGTA, mercaptoethanol4.7H2O, NADH, ATP, the reagent one that glyceraldehyde 3-phosphate dehydrogenase mixes, the reagent two being made of Tris-HCl；The principle of this method is that 3 glyceraldehyde phosphate dehydrogenase can inversely be catalyzed 1,3 diphosphoglyceric acids and NADH generate 3 glyceraldehyde phosphates, Phos and NAD, 3 glyceraldehyde phosphate dehydrogenase and NADH are added in the reaction system, the reduction amount that NADH is measured at 340nm can reflect the height of 1,3 diphospho glycerate acid contents.The features such as present invention has applied widely, and easy to operate, detection time is short, and detection sensitivity is high, low in cost, reproducible, and accuracy rate is high.
Description
Technical field
The invention belongs to life science fields, and in particular to a kind of 1, the 3- diphosphoglyceric acid based on micromethod contains
Measure assay kit and its method.
Background technique
1,3 diphosphoglyceric acid is the mesostate of glycolytic cycle, with gluconeogenesis approach, internal blood sugar concentration
It remains closely related with the generation of diabetes, plays a significant role in body sugar, rouge, protein metabolism disorders.
The method of 1,3 diphospho glycerate acid contents of existing detection is one step sandwich ELISA method of double antibody at present
(ELISA), but this method that there are detection cycles is long, at high cost, narrow application range, stability is poor, and accuracy is low and false positive
The disadvantages of, this causes at present, and there are no a kind of examinations that 1,3 diphospho glycerate acid contents are effectively detected using biochemical method on the market
Agent box and its detection method.
Summary of the invention
In order to overcome the drawbacks of the prior art, the present invention is intended to provide a kind of 1,3- diphosphoglyceric acid based on micromethod
Assay kit and its method, have applied widely, and easy to operate, detection time is short, and detection sensitivity is high, at low cost
It is honest and clean, reproducible, the features such as accuracy rate is high.
To realize above-mentioned technical purpose and the technique effect, the invention is realized by the following technical scheme:
A kind of 1,3- diphosphoglyceric acid assay kit based on micromethod, including following reagent:
Extracting solution, liquid 100mL × 1 bottle, by Tris-HCl, EGTA, mercaptoethanol, glycerol is mixed, as 100mL capacity
In bottle, 4 DEG C of preservations；
Reagent one, pulvis × 1 bottle, by cysteine, MgSO4.7H2O, NADH, ATP, glyceraldehyde 3-phosphate dehydrogenase mixing and
At, be placed in 30mL reagent bottle, -20 DEG C preservation；
Reagent two, liquid 20mL × 1 bottle, is made of Tris-HCl, is placed in 20mL volumetric flask, 4 DEG C of preservations.
Further, the concrete component contained in the extracting solution is 100mL 100mM pH8.2 Tris-HCl, 38mg
EGTA, 50 μ L mercaptoethanols and 5mL glycerol.
Further, the concrete component contained in the reagent one is 8.4mg cysteine, 8.4mg MgSO4.7H2O,
2.4mg NADH, 7.2mg ATP and 0.5mg glyceraldehyde 3-phosphate dehydrogenase.
Further, the concrete component contained in the reagent two is 84mM pH7.6 Tris-HCl.
A kind of 1,3- diphosphoglyceric acid content assaying method based on micromethod using mentioned reagent box, specific steps
It is as follows:
The preparation of step 1 instrument and articles；
Prepare spectrophotometer or microplate reader, thermostat water bath, desk centrifuge, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The extraction of 1,3 diphosphoglyceric acid of step 2；
Animal vegetable tissue: weighing 0.1g sample, and extracting solution described in 1mL is added, and ice bath homogenate, it is 8000g that eccentricity, which is then arranged,
It is centrifuged 10min at 4 DEG C, supernatant is taken to measure；
Bacterium or culture cell: it first collects in bacterium or cell to centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell
Quantity (104It is a): extracting liquid volume (mL) is (500 ~ 1000): 1 ratio, ultrasonic disruption bacterium or cell；Finally it is arranged
Eccentricity is 8000g, is centrifuged 10min at 4 DEG C, takes supernatant, is set to be measured on ice；
The preheating and adjusting of step 3 instrument；
Spectrophotometer or microplate reader preheat 30min or more, adjusting wavelength to 340nm, distilled water zeroing；
The measurement of 1,3 diphospho glycerate acid content of step 4 operates；
The preparation of working solution: the reagent two is all poured into the reagent bottle of the reagent one, is sufficiently dissolved, 37 DEG C or 25 DEG C
Preheating 10 minutes；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
The working solution that 10 μ L samples and 190 μ L are prepared is added in micro quartz colorimetric utensil or 96 orifice plates, mixes, record
The light absorption value A2 after light absorption value A1 and 5min20s at 340nm when 20s calculates Δ A=A1-A2；
The calculating of 1,3 diphospho glycerate acid content of step 5；
A. the calculation formula measured with micro quartz colorimetric utensil is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=643 ÷ ×
ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × V sample ÷ VSample is total) T=643 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷
T=1.286×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=1cm indicates cuvette optical path；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells；
B. the calculation formula measured with 96 orifice plates is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=1286 ÷
×ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × VSample÷VSample is total) T=1286 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷ T
=2.573×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=0.5cm indicates 96 orifice plate optical paths；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells.
Further, in step 2, the quantity for collecting bacterium or cell is 5,000,000, and the volume that the extracting solution is added is
1mL。
Further, in step 2, the specific method of ultrasonication bacterium or cell is the setting ultrasound under condition of ice bath
Power 20% or 200W, ultrasonication 3s, interval 10s, repetitive cycling 30 times.
Further, in step 4, the preheating temperature of the working solution for mammalian sample detection is 37 DEG C, for removing
The preheating temperature of the working solution of other species pattern detections other than mammal is 25 DEG C.
Further, the lowest detection line of 1,3- diphosphoglyceric acid content assaying method of the invention is 0.1nmol/mg
prot。
Measuring principle of the invention is that 3 glyceraldehyde phosphate dehydrogenase can inversely be catalyzed 1,3 diphosphoglyceric acids and NADH is raw
At 3 glyceraldehyde phosphates, Phos and NAD, 3 glyceraldehyde phosphate dehydrogenase and NADH are added in the reaction system, is measured at 340nm
The reduction amount of NADH can reflect the height of 1,3 diphospho glycerate acid contents.At present it is not yet found that document is used with having invention
The method detects 1,3 diphosphonic acid contents.
Compared with prior art, the beneficial effects of the present invention are:
1, detection method of the invention is suitable for the various samples such as animal, plant, microorganism and culture cell, only fits with ELISA
It is compared for mammal, there is significant advantage.
2, detection method lowest detection of the invention is limited to 0.1 nmol/mg prot, substantially increases detection sensitivity.
3, detection method of the invention is easy to operate, it is only necessary to the measurement of a sample, and this method can be completed within 5 minutes
By simply preparing working solution, two kinds of reagents of sample and working solution only need to be added in 96 orifice plates can be completed measurement, detect and
It is convenient, and detection time is greatly saved.
4, the reagent low in raw material price of detection method of the invention, cost are extremely low.
5, preferably, the coefficient of variation (CV value) is within 2% for the repeatability of detection method of the invention.
6, the accuracy of detection method of the invention is higher, sample recovery rate 98%-102%.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And can be implemented in accordance with the contents of the specification, it is described in detail below with presently preferred embodiments of the present invention.Specific reality of the invention
Mode is applied to be shown in detail by following embodiment.
Specific embodiment
Below in conjunction with embodiment, next the present invention will be described in detail.
A kind of 1,3- diphosphoglyceric acid assay kit based on micromethod, including following reagent:
Extracting solution, liquid 100mL × 1 bottle, by 100mL 100mM pH8.2 Tris-HCl, 38mg EGTA, 50 μ L sulfydryl second
Alcohol, 5mL glycerol mix, and as in 100mL volumetric flask, 4 DEG C are saved；
Reagent one, pulvis × 1 bottle, by 8.4mg cysteine, 8.4mg MgSO4.7H2O, 2.4mg NADH, 7.2mg ATP,
0.5mg glyceraldehyde 3-phosphate dehydrogenase mixes, and is placed in 30mL reagent bottle, -20 DEG C of preservations；
Reagent two, liquid 20mL × 1 bottle are made of 84mM pH7.6 Tris-HCl, are placed in 20mL volumetric flask, 4 DEG C of preservations.
A kind of 1,3- diphosphoglyceric acid content assaying method based on micromethod using mentioned reagent box, specific steps
It is as follows:
The preparation of step 1 instrument and articles；
Prepare spectrophotometer or microplate reader, thermostat water bath, desk centrifuge, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The extraction of 1,3 diphosphoglyceric acid of step 2；
Animal vegetable tissue: weighing 0.1g sample, and extracting solution described in 1mL is added, and ice bath homogenate, it is 8000g that eccentricity, which is then arranged,
It is centrifuged 10min at 4 DEG C, supernatant is taken to measure；
Bacterium or culture cell: it first collects in bacterium or cell to centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell
Quantity (104It is a): extracting liquid volume (mL) is (500 ~ 1000): 1 ratio (is extracted it is recommended that 1mL is added in 5,000,000 bacteriums or cell
Liquid), ultrasonic disruption bacterium or cell (ice bath, power 20% or 200W, ultrasonication 3s are spaced 10s, repeat 30 times)；Most
Setting eccentricity is 8000g afterwards, is centrifuged 10min at 4 DEG C, takes supernatant, sets to be measured on ice；
The preheating and adjusting of step 3 instrument；
Spectrophotometer or microplate reader preheat 30min or more, adjusting wavelength to 340nm, distilled water zeroing；
The measurement of 1,3 diphospho glycerate acid content of step 4 operates；
The preparation of working solution: the reagent two is all poured into the reagent bottle of the reagent one, is sufficiently dissolved, 37 DEG C of (lactations
Animal) or 25 DEG C (other species) preheat 10 minutes；- 20 DEG C of preservations after exhaustless reagent packing, forbid multigelation；
The working solution that 10 μ L samples and 190 μ L are prepared is added in micro quartz colorimetric utensil or 96 orifice plates, mixes, record
The light absorption value A2 after light absorption value A1 and 5min20s at 340nm when 20s calculates Δ A=A1-A2；
The calculating of 1,3 diphospho glycerate acid content of step 5；
A. the calculation formula measured with micro quartz colorimetric utensil is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=643 ÷ ×
ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × V sample ÷ VSample is total) T=643 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷
T=1.286×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=1cm indicates cuvette optical path；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells；
B. the calculation formula measured with 96 orifice plates is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=1286 ÷
×ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × VSample÷VSample is total) T=1286 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷ T
=2.573×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=0.5cm indicates 96 orifice plate optical paths；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells.
The lowest detection line of 1,3 diphospho glycerate acid content measuring method of the invention is 0.1nmol/mg prot.
Simply to illustrate that technical concepts and features of the invention, its purpose is allows in the art above-described embodiment
Those of ordinary skill cans understand the content of the present invention and implement it accordingly, and it is not intended to limit the scope of the present invention.It is all
It is changes or modifications equivalent made by the essence of content according to the present invention, should be covered by the scope of protection of the present invention.
Claims (9)
1. 1, the 3- diphosphoglyceric acid assay kit based on micromethod, which is characterized in that including following reagent:
Extracting solution, liquid 100mL × 1 bottle, by Tris-HCl, EGTA, mercaptoethanol, glycerol is mixed, as 100mL capacity
In bottle, 4 DEG C of preservations；
Reagent one, pulvis × 1 bottle, by cysteine, MgSO4.7H2O, NADH, ATP, glyceraldehyde 3-phosphate dehydrogenase mixing and
At, be placed in 30mL reagent bottle, -20 DEG C preservation；
Reagent two, liquid 20mL × 1 bottle, is made of Tris-HCl, is placed in 20mL volumetric flask, 4 DEG C of preservations.
2. 1, the 3- diphosphoglyceric acid assay kit according to claim 1 based on micromethod, feature exist
In: the concrete component contained in the extracting solution is 100mL 100mM pH8.2 Tris-HCl, 38mg EGTA, 50 μ L sulfydryls
Ethyl alcohol and 5mL glycerol.
3. 1, the 3- diphosphoglyceric acid assay kit according to claim 1 based on micromethod, feature exist
In: the concrete component contained in the reagent one is 8.4mg cysteine, 8.4mg MgSO4.7H2O, 2.4mg NADH, 7.2mg
ATP and 0.5mg glyceraldehyde 3-phosphate dehydrogenase.
4. 1, the 3- diphosphoglyceric acid assay kit according to claim 1 based on micromethod, feature exist
In: the concrete component contained in the reagent two is 84mM pH7.6 Tris-HCl.
5. using 1, the 3- diphosphoglyceric acid content assaying method based on micromethod of kit as described in claim 1,
It is characterized in that, specific step is as follows:
The preparation of step 1 instrument and articles；
Prepare spectrophotometer or microplate reader, thermostat water bath, desk centrifuge, adjustable pipette, micro quartz colorimetric utensil
Or 96 orifice plates, mortar, ice and distilled water；
The extraction of 1,3 diphosphoglyceric acid of step 2；
Animal vegetable tissue: weighing 0.1g sample, and extracting solution described in 1mL is added, and ice bath homogenate, it is 8000g that eccentricity, which is then arranged,
It is centrifuged 10min at 4 DEG C, supernatant is taken to measure；
Bacterium or culture cell: it first collects in bacterium or cell to centrifuge tube, supernatant is abandoned after centrifugation；Then according to bacterium or cell
Quantity (104It is a): extracting liquid volume (mL) is (500 ~ 1000): 1 ratio, ultrasonic disruption bacterium or cell；Finally it is arranged
Eccentricity is 8000g, is centrifuged 10min at 4 DEG C, takes supernatant, is set to be measured on ice；
The preheating and adjusting of step 3 instrument；
Spectrophotometer or microplate reader preheat 30min or more, adjusting wavelength to 340nm, distilled water zeroing；
The measurement of 1,3 diphospho glycerate acid content of step 4 operates；
The preparation of working solution: the reagent two is all poured into the reagent bottle of the reagent one, sufficiently dissolves, preheats 10 points
Clock；
The working solution that 10 μ L samples and 190 μ L are prepared is added in micro quartz colorimetric utensil or 96 orifice plates, mixes, record
The light absorption value A2 after light absorption value A1 and 5min20s at 340nm when 20s calculates Δ A=A1-A2；
The calculating of 1,3 diphospho glycerate acid content of step 5；
A. the calculation formula measured with micro quartz colorimetric utensil is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=643 ÷ ×
ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × V sample ÷ VSample is total) T=643 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷ T
=1.286×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=1cm indicates cuvette optical path；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells；
B. the calculation formula measured with 96 orifice plates is as follows:
1) it is calculated by sample protein concentration；
1,3 diphosphoglyceric acid (nmol/mg prot)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (VSample× Cpr) T=1286 ÷ ×
ΔA÷Cpr；
2) it is calculated by sample fresh weight；
1,3 diphosphoglyceric acid (nmol/g fresh weight)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (W × VSample÷VSample is total) T=1286 ÷
×ΔA÷W；
3) it is calculated by bacterium or cell density；
1,3 diphosphoglyceric acid (nmol/104Cell)=[Δ A × VIt is anti-total÷ (ε × d) × 109] ÷ (500 × VSample÷VSample is total) ÷ T=
2.573×ΔA；
Wherein, VIt is anti-total=2×10-4L indicates reaction system total volume；
ε=6.22×103L/mol/cm indicates NADH molar extinction coefficient；
D=0.5cm indicates 96 orifice plate optical paths；
VSample=0.01mL indicates the volume that sample is added；
VSample is total=1mL indicates the volume that extracting solution is added；
T=5min indicates the reaction time；
Cpr indicates sample protein concentration, unit mg/mL；
W indicates sample quality, unit g；
500 indicate 5,000,000 bacteriums or total number of cells.
6. 1, the 3- diphosphoglyceric acid content assaying method according to claim 5 based on micromethod, it is characterised in that:
In step 2, the quantity for collecting bacterium or cell is 5,000,000, and the volume that the extracting solution is added is 1mL.
7. 1, the 3- diphosphoglyceric acid content assaying method according to claim 5 based on micromethod, it is characterised in that:
In step 2, the specific method of ultrasonication bacterium or cell is that ultrasonic power 20% or 200W, ultrasound is arranged under condition of ice bath
Broken 3s, interval 10s, repetitive cycling 30 times.
8. 1, the 3- diphosphoglyceric acid content assaying method according to claim 5 based on micromethod, it is characterised in that:
In step 4, the preheating temperature of the working solution for mammalian sample detection is 37 DEG C, for other in addition to mammal
The preheating temperature of the working solution of species pattern detection is 25 DEG C.
9. 1, the 3- diphosphoglyceric acid content assaying method according to claim 5 based on micromethod, it is characterised in that:
The lowest detection line of the 1,3 diphospho glycerate acid content measuring method is 0.1nmol/mg prot.
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| CN110779887A (en) * | 2019-09-30 | 2020-02-11 | 浙江大学 | Method for determining phosphoglycerate kinase activity |
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