CN105648034A - Kit for determining NADP phosphatase activity and determination method thereof - Google Patents

Kit for determining NADP phosphatase activity and determination method thereof Download PDF

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CN105648034A
CN105648034A CN201511013813.5A CN201511013813A CN105648034A CN 105648034 A CN105648034 A CN 105648034A CN 201511013813 A CN201511013813 A CN 201511013813A CN 105648034 A CN105648034 A CN 105648034A
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reagent
pipe
bottle
phosphorus
nadp
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赵林川
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SUZHOU COMIN BIOTECHNOLOGY Co Ltd
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SUZHOU COMIN BIOTECHNOLOGY Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

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Abstract

The invention discloses a kit for determining NADP phosphatase activity and a determination method thereof. The kit comprises the following reagents: a bottle of extract, a bottle of reagent which is prepared by mixing Tris which is dissolved in water and glacial acetic acid, five tubes of powder which is composed of NADP, a bottle of reagent which is composed of ascorbic acid, a bottle of powder which is composed of ammonium molybdate, a bottle of liquid which is mixed from concentrated sulfuric acid and water, and a bottle of 10mmol/L standard phosphor stock solution. The method is based on spectrophotometry, and NADP phosphatase activity is indirectly determined by a phosphor determination method with ammonium molybdate; compared with a phosphor determination method with malachite green, the determination range is wider; a light absorption value of a determination tube and a light absorption value of a standard tube are compared, and the NADP phosphatase activity is calculated; the method is suitable for determining activities of different NADP phosphatase, and dilution of samples is not needed. Reagents used in the kit are common reagents with extremely low costs. The kit has the advantages of detection convenience and rapid detection, the kit can be used for determining multiple samples simultaneously, and the repeatability in 24 hours after color developing is good.

Description

A kind of NADP phosphatase activity measures test kit and method thereof
Technical field
The invention belongs to life science field, relate to a kind of test kit, be specifically related to a kind of NADP phosphatase activity and measure test kit and method thereof.
Background technology
NADP phosphatase is primarily present in plant tissue, is unique catalysis NADP in organism+It is degraded to NAD+Enzyme, regulate and control the balance between NAD and NADP together with NADK.
The assay method of existing two kinds of NADP phosphatase activities at present, one is to be changed by alcohol dehydrogenase Enzymatic cycling assay NAD, and another kind is to measure NAD change by peacock green fixing phosphorus method. Needing the strict temperature and time controlling reaction by the method for alcohol dehydrogenase Enzymatic cycling assay NAD change, reaction condition is wayward, and reagent cost is higher. Peacock green fixing phosphorus method is applicable to measure phosphorus concentration sample in 0.06-2 �� g range, if the phosphorus content that in sample, NADP phosphatase catalytic produces is too high, measures after needing dilution. And the process that measures needs to do preliminary experiment, to determine whether the Phos that in sample, NADP phosphatase activity produces has exceeded measurement range, relatively complicated.
Summary of the invention
In order to overcome the defect of prior art, it is desirable to provide a kind of NADP phosphatase activity measures test kit and method thereof, NADP phosphatase activity can be calculated fast and accurately.
For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the present invention is achieved through the following technical solutions:
A kind of NADP phosphatase activity measures test kit, including following reagent:
Extracting solution, 1 bottle, 4 DEG C of preservations, sucrose, EDTA, bovine serum albumin and Phenylmethanesulfonyl fluoride formulated after being dissolved in Tris-HCl buffer solution, it is placed in 60mL reagent bottle;
Reagent one, liquid �� 1 bottle, 4 DEG C of preservations, Tris formulated after being dissolved in the water mixed liquor with glacial acetic acid, it is placed in 30mL reagent bottle;
Reagent two, powder �� 5 ,-20 DEG C of preservations, form by NADP, be placed in 1mL centrifuge tube;
Reagent three, powder �� 1 bottle, 4 DEG C of preservations, it is made up of ascorbic acid, is placed in 25mL reagent bottle;
Reagent four, powder �� 1 bottle, 4 DEG C of preservations, it is made up of ammonium molybdate, is placed in 25mL reagent bottle;
Reagent five, liquid �� 1 bottle, room temperature preservation, concentrated sulphuric acid and water mix, be placed in 25mL reagent bottle;
Reagent six, 10mmol/L standard phosphorus stock solution �� 1 bottle, 4 DEG C of preservations, by Na2HPO4��12H2O is formulated after being dissolved in water, is placed in 10mL reagent bottle.
Further, in described extracting solution, the concentration of Tris-HCl buffer solution is 50mmol/L, pH=7.4, and volume is 60mL, and the quality of sucrose is the quality of 5.1g, EDTA is 52.6mg, and the quality of bovine serum albumin is 0.3g, and the quality of Phenylmethanesulfonyl fluoride is 1mg;
Further, in described reagent one, the quality of Tris is the pH=5.5 that 0.182g, Tris are dissolved in after water with the mixed liquor of glacial acetic acid, and volume is 30mL;
Further, in described reagent two, the quality of the NADP in every centrifuge tube is 5.5mg;
Further, in described reagent three, the quality of ascorbic acid is 2.5g;
Further, in described reagent four, the quality of ammonium molybdate is 0.625g;
Further, in described reagent five, the volume of concentrated sulphuric acid is 3.47mL, and the volume of water is 21.53mL;
Further, in described reagent six, Na2HPO4��12H2The quality of O is 35.814mg, and the volume of water is 10mL.
NADP phosphatase catalytic NADP hydrolysis generates NAD and Phos, can indirectly reflect the height of NADP phosphatase activity by measuring the change of NAD or Phos.
A kind of NADP phosphatase activity assay method based on spectrophotography, comprises the following steps:
The preparation of step 1 instrument and articles for use;
Prepare visible spectrophotometer, thermostat water bath, desk centrifuge, adjustable pipette, 1mL glass cuvette, mortar, ice and distilled water;
The pre-treatment of step 2 sample;
According to liver mass (g): extracting liquid volume (mL) is 1:(5 ~ 10) ratio, carry out ice bath homogenate, then adopt eccentricity 8000g, at 4 DEG C, centrifugal 10min, take supernatant, put to be measured on ice;
The preparation before use of step 3 reagent;
1) reagent two adds reagent one described in 1mL every described, fully dissolve standby, now with the current;
2) in described reagent three, 25mL distilled water is added, standby after dissolving;
3) in described reagent four, 25mL distilled water is added, standby after dissolving;
Step 40.5 ��m ol/mL standard phosphorus application liquid is prepared:
Described reagent six is carried out 20 times of dilutions, namely takes reagent six described in 0.5mL and add 9.5mL distilled water, fully mix;
Step 5 determines the preparation of phosphorus reagent;
By water: reagent three: reagent four: the proportions of reagent five=2:1:1:1, the phosphorus reagent of determining prepared should be light yellow, if colourless, reagent lost efficacy, if blueness is then polluted for phosphorus, determined phosphorus agent matching while using;
Step 6 enzymatic reaction;
1) in mensuration pipe and control tube, all add the described reagent one of 300 �� L and the described reagent two of 100 �� L, and under 37 DEG C (mammals) or 25 DEG C (other species), all preheat 5min;
2) in described mensuration pipe, add the sample of pre-treatment in 100 �� L steps 2, described control tube adds 100 �� L distilled water;
3) by described mensuration pipe accurate response 30min under 37 DEG C (mammals) or 25 DEG C (other species), then boiling water bath 5min(covers tightly, to prevent moisture loss), after cooling, adopting eccentricity 10000g again, under room temperature, centrifugal 5min, finally takes supernatant;
Step 7 determines phosphorus;
1) what add 0.5 ��m of ol/mL standard phosphorus application liquid of 100 �� L and 1000 �� L in standard pipe determines phosphorus reagent, what add the distilled water of 100 �� L and 1000 �� L in blank tube determines phosphorus reagent, measure in pipe add the supernatant extracted in 100 �� L steps 6 and 1000 �� L determine phosphorus reagent, what add the supernatant extracted in 100 �� L steps 6 and 1000 �� L in control tube determines phosphorus reagent;
2) material in each pipe is mixed, difference water-bath 30min under 37 DEG C (mammals) or 25 DEG C (other species);
3) then each pipe being cooled to room temperature, then by spectrophotometric wavelength regulation at 660nm place, distilled water returns to zero, and records each pipe light absorption value;
Step 8, according to each pipe light absorption value drawn in step 7, calculates NADP phosphatase activity;
1) calculate by Tissue protein concentration:
Definition: the amount of every milligram of histone NADP phosphatase decomposition NADP 1 ��m of ol Phos of generation is a NADP Phosphatase Activity unit per hour;
NADP phosphatase (U/mgprot)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube)��(VSample��Cpr)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� Cpr;
2) calculate by sample fresh weight:
Definition: every g organizes the amount that NADP phosphatase decomposes NADP 1 ��m of ol Phos of generation to be a NADP Phosphatase Activity unit per hour;
NADP phosphatase (U/g fresh weight)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� VAlways��(W��VSample��VSample is total)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� W;
Wherein, A represents the light absorption value of each pipe in step 7;
CStandard pipe=0.5 ��m of ol/mL, represents the concentration of standard pipe;
VAlways=0.5mL, represents the cumulative volume of enzymatic reaction;
VSample=0.1mL, represents the volume adding sample;
VSample is total=1mL, represents the volume adding extracting solution;
T=0.5h, represents the response time;
Cpr represents the concentration of sample protein, and unit is mg/mL;
W represents the fresh weight of sample, and unit is g.
The invention has the beneficial effects as follows:
The method of the present invention is based on spectrophotography, and carry out indirect determination NADP phosphatase activity by ammonium molybdate fixing phosphorus method, compare peacock green fixing phosphorus method, measurement range is more extensive, measure pipe light absorption value and standard pipe light absorption value comparison can calculate NADP phosphatase activity, it is applicable to measure the sample of different NADP phosphatase activity, therefore sample need not be diluted. The test kit agents useful for same of the present invention is common agents, and cost is extremely low. The test kit of the present invention is not only easy to detect, quickly, and can measure multiple sample simultaneously, develops the color in latter 24 hours, and repeatability is better.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, and can be practiced according to the content of description, describes in detail with presently preferred embodiments of the present invention below. The specific embodiment of the present invention is shown in detail in by following example.
Detailed description of the invention
Below in conjunction with embodiment, describe the present invention in detail.
Embodiment 1
A kind of NADP phosphatase activity measures test kit, including following reagent:
Extracting solution, 1 bottle, 4 DEG C of preservations, 5.1g sucrose, 52.6mgEDTA, 0.3g bovine serum albumin and 1mg Phenylmethanesulfonyl fluoride being dissolved in concentration is 50mmol/L, pH=7.4, volume be 60mL Tris-HCl buffer solution after formulated, be placed in 60mL reagent bottle;
Reagent one, liquid �� 1 bottle, 4 DEG C of preservations, 0.182gTris regulate pH=5.5 with glacial acetic acid after being dissolved in a small amount of water, be finally settled to 30mL with water formulated, be placed in 30mL reagent bottle;
Reagent two, powder �� 5 ,-20 DEG C of preservations, form by 5.5mgNADP, be placed in 1mL centrifuge tube;
Reagent three, powder �� 1 bottle, 4 DEG C of preservations, it is made up of 2.5g ascorbic acid, is placed in 25mL reagent bottle;
Reagent four, powder �� 1 bottle, 4 DEG C of preservations, it is made up of 0.625g ammonium molybdate, is placed in 25mL reagent bottle;
Reagent five, liquid �� 1 bottle, room temperature preservation, 3.47mL concentrated sulphuric acid and 21.53mL water mix, be placed in 25mL reagent bottle;
Reagent six, 10mmol/L standard phosphorus stock solution �� 1 bottle, 4 DEG C of preservations, by 35.814mgNa2HPO4��12H2O is formulated after being dissolved in 10mL water, is placed in 10mL reagent bottle.
A kind of based on spectrophotography and utilize mentioned reagent box measure NADP phosphatase activity method, comprise the following steps:
The preparation of step 1 instrument and articles for use;
Prepare visible spectrophotometer, thermostat water bath, desk centrifuge, adjustable pipette, 1mL glass cuvette, mortar, ice and distilled water;
The pre-treatment of step 2 sample;
According to liver mass (g): extracting liquid volume (mL) is 1:(5 ~ 10) ratio (suggestion weigh about 0.1g tissue, add 1mL extracting solution), carry out ice bath homogenate, adopt eccentricity 8000g again, at 4 DEG C, centrifugal 10min, take supernatant, put to be measured on ice;
The preparation before use of step 3 reagent;
1) reagent two adds reagent one described in 1mL every described, fully dissolve standby, now with the current;
2) in described reagent three, 25mL distilled water is added, standby after dissolving;
3) in described reagent four, 25mL distilled water is added, standby after dissolving;
Step 40.5 ��m ol/mL standard phosphorus application liquid is prepared:
Described reagent six is carried out 20 times of dilutions, namely takes reagent six described in 0.5mL and add 9.5mL distilled water, fully mix;
Step 5 determines the preparation of phosphorus reagent;
By water: reagent three: reagent four: the proportions of reagent five=2:1:1:1, the phosphorus reagent of determining prepared should be light yellow, if colourless, reagent lost efficacy, if blueness is then polluted for phosphorus, determined phosphorus agent matching while using;
Step 6 enzymatic reaction;
1) in measuring pipe and control tube, all add the described reagent one of 300 �� L and the described reagent two of 100 �� L, and under 37 DEG C (mammals) or 25 DEG C (other species), all preheat 5min;
2) in described mensuration pipe, add the sample of pre-treatment in 100 �� L steps 2, described control tube adds 100 �� L distilled water;
3) by described mensuration pipe accurate response 30min under 37 DEG C (mammals) or 25 DEG C (other species), then boiling water bath 5min(covers tightly, to prevent moisture loss), after cooling, adopting eccentricity 10000g again, under room temperature, centrifugal 5min, finally takes supernatant;
Step 7 determines phosphorus;
1) what add 0.5 ��m of ol/mL standard phosphorus application liquid of 100 �� L and 1000 �� L in standard pipe determines phosphorus reagent, what add the distilled water of 100 �� L and 1000 �� L in blank tube determines phosphorus reagent, measure in pipe add the supernatant extracted in 100 �� L steps 6 and 1000 �� L determine phosphorus reagent, what add the supernatant extracted in 100 �� L steps 6 and 1000 �� L in control tube determines phosphorus reagent;
2) material in each pipe is mixed, difference water-bath 30min under 37 DEG C (mammals) or 25 DEG C (other species);
3) then each pipe being cooled to room temperature, then by spectrophotometric wavelength regulation at 660nm place, distilled water returns to zero, and records each pipe light absorption value;
Step 8 is according to each pipe light absorption value drawn in step 7, and presses Tissue protein concentration, calculates NADP phosphatase activity and calculates;
Definition: the amount of every milligram of histone NADP phosphatase decomposition NADP 1 ��m of ol Phos of generation is a NADP Phosphatase Activity unit per hour;
NADP phosphatase (U/mgprot)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube)��(VSample��Cpr)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� Cpr;
Wherein, A represents the light absorption value of each pipe in step 7;
CStandard pipe=0.5 ��m of ol/mL, represents the concentration of standard pipe;
VAlways=0.5mL, represents the cumulative volume of enzymatic reaction;
VSample=0.1mL, represents the volume adding sample;
T=0.5h, represents the response time;
Cpr represents the concentration of sample protein, and unit is mg/mL.
Embodiment 2
A kind of NADP phosphatase activity measures test kit, including following reagent:
Extracting solution, 1 bottle, 4 DEG C of preservations, 5.1g sucrose, 52.6mgEDTA, 0.3g bovine serum albumin and 1mg Phenylmethanesulfonyl fluoride being dissolved in concentration is 50mmol/L, pH=7.4, volume be 60mL Tris-HCl buffer solution after formulated, be placed in 60mL reagent bottle;
Reagent one, liquid �� 1 bottle, 4 DEG C of preservations, 0.182gTris regulate pH=5.5 with glacial acetic acid after being dissolved in a small amount of water, be finally settled to 30mL with water formulated, be placed in 30mL reagent bottle;
Reagent two, powder �� 5 ,-20 DEG C of preservations, form by 5.5mgNADP, be placed in 1mL centrifuge tube;
Reagent three, powder �� 1 bottle, 4 DEG C of preservations, it is made up of 2.5g ascorbic acid, is placed in 25mL reagent bottle;
Reagent four, powder �� 1 bottle, 4 DEG C of preservations, it is made up of 0.625g ammonium molybdate, is placed in 25mL reagent bottle;
Reagent five, liquid �� 1 bottle, room temperature preservation, 3.47mL concentrated sulphuric acid and 21.53mL water mix, be placed in 25mL reagent bottle;
Reagent six, 10mmol/L standard phosphorus stock solution �� 1 bottle, 4 DEG C of preservations, by 35.814mgNa2HPO4��12H2O is formulated after being dissolved in 10mL water, is placed in 10mL reagent bottle.
A kind of based on spectrophotography and utilize mentioned reagent box measure NADP phosphatase activity method, comprise the following steps:
The preparation of step 1 instrument and articles for use;
Prepare visible spectrophotometer, thermostat water bath, desk centrifuge, adjustable pipette, 1mL glass cuvette, mortar, ice and distilled water;
The pre-treatment of step 2 sample;
According to liver mass (g): extracting liquid volume (mL) is 1:(5 ~ 10) ratio (suggestion weigh about 0.1g tissue, add 1mL extracting solution), carry out ice bath homogenate, adopt eccentricity 8000g again, at 4 DEG C, centrifugal 10min, take supernatant, put to be measured on ice;
The preparation before use of step 3 reagent;
1) reagent two adds reagent one described in 1mL every described, fully dissolve standby, now with the current;
2) in described reagent three, 25mL distilled water is added, standby after dissolving;
3) in described reagent four, 25mL distilled water is added, standby after dissolving;
Step 40.5 ��m ol/mL standard phosphorus application liquid is prepared:
Described reagent six is carried out 20 times of dilutions, namely takes reagent six described in 0.5mL and add 9.5mL distilled water, fully mix;
Step 5 determines the preparation of phosphorus reagent;
By water: reagent three: reagent four: the proportions of reagent five=2:1:1:1, the phosphorus reagent of determining prepared should be light yellow, if colourless, reagent lost efficacy, if blueness is then polluted for phosphorus, determined phosphorus agent matching while using;
Step 6 enzymatic reaction;
1) in measuring pipe and control tube, all add the described reagent one of 300 �� L and the described reagent two of 100 �� L, and under 37 DEG C (mammals) or 25 DEG C (other species), all preheat 5min;
2) in described mensuration pipe, add the sample of pre-treatment in 100 �� L steps 2, described control tube adds 100 �� L distilled water;
3) by described mensuration pipe accurate response 30min under 37 DEG C (mammals) or 25 DEG C (other species), then boiling water bath 5min(covers tightly, to prevent moisture loss), after cooling, adopting eccentricity 10000g again, under room temperature, centrifugal 5min, finally takes supernatant;
Step 7 determines phosphorus;
1) what add 0.5 ��m of ol/mL standard phosphorus application liquid of 100 �� L and 1000 �� L in standard pipe determines phosphorus reagent, what add the distilled water of 100 �� L and 1000 �� L in blank tube determines phosphorus reagent, measure in pipe add the supernatant extracted in 100 �� L steps 6 and 1000 �� L determine phosphorus reagent, what add the supernatant extracted in 100 �� L steps 6 and 1000 �� L in control tube determines phosphorus reagent;
2) material in each pipe is mixed, difference water-bath 30min under 37 DEG C (mammals) or 25 DEG C (other species);
3) then each pipe being cooled to room temperature, then by spectrophotometric wavelength regulation at 660nm place, distilled water returns to zero, and records each pipe light absorption value;
Step 8 is according to each pipe light absorption value drawn in step 7, by sample fresh weight, calculates NADP phosphatase activity;
Definition: every g organizes the amount that NADP phosphatase decomposes NADP 1 ��m of ol Phos of generation to be a NADP Phosphatase Activity unit per hour;
NADP phosphatase (U/g fresh weight)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� VAlways��(W��VSample��VSample is total)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� W;
Wherein, A represents the light absorption value of each pipe in step 7;
CStandard pipe=0.5 ��m of ol/mL, represents the concentration of standard pipe;
VAlways=0.5mL, represents the cumulative volume of enzymatic reaction;
VSample=0.1mL, represents the volume adding sample;
VSample is total=1mL, represents the volume adding extracting solution;
T=0.5h, represents the response time;
Cpr represents the concentration of sample protein, and unit is mg/mL;
W represents the fresh weight of sample, and unit is g.
Above-described embodiment, simply to illustrate that the technology of the present invention is conceived and feature, its objective is to be in that to allow one of ordinary skilled in the art will appreciate that present disclosure and to implement according to this, can not limit the scope of the invention with this. The change of every equivalence done by the essence of present invention or modification, all should be encompassed in protection scope of the present invention.

Claims (3)

1. a NADP phosphatase activity measures test kit, it is characterised in that include following reagent:
Extracting solution, 1 bottle, 4 DEG C of preservations, sucrose, EDTA, bovine serum albumin and Phenylmethanesulfonyl fluoride formulated after being dissolved in Tris-HCl buffer solution, it is placed in 60mL reagent bottle;
Reagent one, liquid �� 1 bottle, 4 DEG C of preservations, Tris formulated after being dissolved in the water mixed liquor with glacial acetic acid, it is placed in 30mL reagent bottle;
Reagent two, powder �� 5 ,-20 DEG C of preservations, form by NADP, be placed in 1mL centrifuge tube;
Reagent three, powder �� 1 bottle, 4 DEG C of preservations, it is made up of ascorbic acid, is placed in 25mL reagent bottle;
Reagent four, powder �� 1 bottle, 4 DEG C of preservations, it is made up of ammonium molybdate, is placed in 25mL reagent bottle;
Reagent five, liquid �� 1 bottle, room temperature preservation, concentrated sulphuric acid and water mix, be placed in 25mL reagent bottle;
Reagent six, 10mmol/L standard phosphorus stock solution �� 1 bottle, 4 DEG C of preservations, by Na2HPO4��12H2O is formulated after being dissolved in water, is placed in 10mL reagent bottle.
2. NADP phosphatase activity according to claim 1 measures test kit, it is characterised in that:
In described extracting solution, the concentration of Tris-HCl buffer solution is 50mmol/L, pH=7.4, and volume is 60mL, and the quality of sucrose is the quality of 5.1g, EDTA is 52.6mg, and the quality of bovine serum albumin is 0.3g, and the quality of Phenylmethanesulfonyl fluoride is 1mg;
In described reagent one, the quality of Tris is the pH=5.5 that 0.182g, Tris are dissolved in after water with the mixed liquor of glacial acetic acid, and volume is 30mL;
In described reagent two, the quality of the NADP in every centrifuge tube is 5.5mg;
In described reagent three, the quality of ascorbic acid is 2.5g;
In described reagent four, the quality of ammonium molybdate is 0.625g;
In described reagent five, the volume of concentrated sulphuric acid is 3.47mL, and the volume of water is 21.53mL;
In described reagent six, Na2HPO4��12H2The quality of O is 35.814mg, and the volume of water is 10mL.
3. the NADP phosphatase activity assay method adopting test kit as claimed in claim 2, it is characterised in that based on spectrophotography, comprise the following steps:
The preparation of step 1 instrument and articles for use;
Prepare visible spectrophotometer, thermostat water bath, desk centrifuge, adjustable pipette, 1mL glass cuvette, mortar, ice and distilled water;
The pre-treatment of step 2 sample;
According to liver mass (g): extracting liquid volume (mL) is 1:(5 ~ 10) ratio, carry out ice bath homogenate, then adopt eccentricity 8000g, at 4 DEG C, centrifugal 10min, take supernatant, put to be measured on ice;
The preparation before use of step 3 reagent;
1) reagent two adds reagent one described in 1mL every described, fully dissolve standby, now with the current;
2) in described reagent three, 25mL distilled water is added, standby after dissolving;
3) in described reagent four, 25mL distilled water is added, standby after dissolving;
Step 40.5 ��m ol/mL standard phosphorus application liquid is prepared:
Described reagent six is carried out 20 times of dilutions, namely takes reagent six described in 0.5mL and add 9.5mL distilled water, fully mix;
Step 5 determines the preparation of phosphorus reagent;
By water: reagent three: reagent four: the proportions of reagent five=2:1:1:1, the phosphorus reagent of determining prepared should be light yellow, if colourless, reagent lost efficacy, if blueness is then polluted for phosphorus, determined phosphorus agent matching while using;
Step 6 enzymatic reaction;
In measuring pipe and control tube, all add the described reagent one of 300 �� L and the described reagent two of 100 �� L, and at 37 DEG C or 25 DEG C, all preheat 5min;
Described mensuration pipe adds the sample of pre-treatment in 100 �� L steps 2, described control tube adds 100 �� L distilled water;
Measuring pipe and control tube all accurate response 30min, then boiling water bath 5min at 37 DEG C or 25 DEG C by described, after cooling, then adopt eccentricity 10000g, under room temperature, centrifugal 5min, finally takes supernatant;
Step 7 determines phosphorus;
1) what add 0.5 ��m of ol/mL standard phosphorus application liquid of 100 �� L and 1000 �� L in standard pipe determines phosphorus reagent, what add the distilled water of 100 �� L and 1000 �� L in blank tube determines phosphorus reagent, measure in pipe add the supernatant extracted in 100 �� L steps 6 and 1000 �� L determine phosphorus reagent, what add the supernatant extracted in 100 �� L steps 6 and 1000 �� L in control tube determines phosphorus reagent;
2) material in each pipe is mixed, difference water-bath 30min at 37 DEG C or 25 DEG C;
3) then each pipe being cooled to room temperature, then by spectrophotometric wavelength regulation at 660nm place, distilled water returns to zero, and records each pipe light absorption value;
Step 8, according to each pipe light absorption value drawn in step 7, calculates NADP phosphatase activity;
1) calculate by Tissue protein concentration:
NADP phosphatase (U/mgprot)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube)��(VSample��Cpr)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� Cpr;
2) calculate by sample fresh weight:
NADP phosphatase (U/g fresh weight)=(CStandard pipe��VAlways)��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� VAlways��(W��VSample��VSample is total)��T=5��(AMeasure pipe-AControl tube)��(AStandard pipe-ABlank tube) �� W;
Wherein, A represents the light absorption value of each pipe in step 7;
CStandard pipe=0.5 ��m of ol/mL, represents the concentration of standard pipe;
VAlways=0.5mL, represents the cumulative volume of enzymatic reaction;
VSample=0.1mL, represents the volume adding sample;
VSample is total=1mL, represents the volume adding extracting solution;
T=0.5h, represents the response time;
Cpr represents the concentration of sample protein, and unit is mg/mL;
W represents the fresh weight of sample, and unit is g.
CN201511013813.5A 2015-12-31 2015-12-31 Kit for determining NADP phosphatase activity and determination method thereof Pending CN105648034A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111721756A (en) * 2019-03-18 2020-09-29 中国农业科学院北京畜牧兽医研究所 Alkaline phosphatase activity detection kit and detection method
CN114235522A (en) * 2021-12-08 2022-03-25 中国科学院东北地理与农业生态研究所 Kit and method for detecting ureide content and allantoic acid content in plant

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* Cited by examiner, † Cited by third party
Title
张申，王杰，高江原主编: "《分子生物学检验技术》", 28 February 2013 *
杨万年，何之常: "孔雀绿定磷法测定植物NADP磷酸酶活性", 《植物生理学通讯》 *
白雪: "湖北麦冬对STZ诱导2型糖尿病小鼠的治疗作用及其作用机理的研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111721756A (en) * 2019-03-18 2020-09-29 中国农业科学院北京畜牧兽医研究所 Alkaline phosphatase activity detection kit and detection method
CN114235522A (en) * 2021-12-08 2022-03-25 中国科学院东北地理与农业生态研究所 Kit and method for detecting ureide content and allantoic acid content in plant
CN114235522B (en) * 2021-12-08 2023-05-02 中国科学院东北地理与农业生态研究所 Kit and method for detecting ureide content and allantoin content in plants

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