CN107782684A - Codehydrogenase Ⅱ NADP+Or NADPH assays kit and its method - Google Patents

Codehydrogenase Ⅱ NADP+Or NADPH assays kit and its method Download PDF

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Publication number
CN107782684A
CN107782684A CN201710928062.2A CN201710928062A CN107782684A CN 107782684 A CN107782684 A CN 107782684A CN 201710928062 A CN201710928062 A CN 201710928062A CN 107782684 A CN107782684 A CN 107782684A
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nadp
nadph
reagent
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eccentricity
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姚金美
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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Abstract

The invention discloses a kind of codehydrogenase Ⅱ NADP+Or NADPH assays kit and its method, its kit include acidic extraction liquid, alkaline extract solution, HEPES and EDTA mixed solution, 6 glucose 1-phosphate1-s, PMS, MTT, 6 glucose phosphate dehydrogenase solution, NaCl solution and the mixed solution of ethanol and distilled water.Its method is to be based on AAS, with NADP in sour, alkaline extract solution extraction sample+And NADPH, NADPH are acted on by PMS hydrogen of passing, and MTT is reduced to first a ceremonial jade-ladle, used in libation, light absorption value is detected under 570nm, determine NADPH contents, 6 glucose phosphate dehydrogenases reduction NADP is recycled+For NADPH, so as to detect NADP+Content.The kit and its assay method of the present invention can effectively determine codehydrogenase Ⅱ NADP+Or NADPH content, there is the features such as selectivity is higher, applied widely, stability is high.

Description

Codehydrogenase Ⅱ NADP+Or NADPH assays kit and its method
Technical field
The invention belongs to life science field, and in particular to a kind of codehydrogenase Ⅱ NADP+Or NADPH assay reagents Box and its method.
Background technology
Codehydrogenase Ⅱ NADP (H) is widely present in animal, plant, microorganism and culture cell, determines NADP+And NADPH Content can calculate NADP（NADPH + NADP+) content and NADPH/NADP+Ratio, it changes and pentose phosphate pathway and life Thing synthesizes and Antioxidation reaction is closely related.NADPH/NADP+Ratio be not only cell redox status outstanding feature it One, and there is important regulating and controlling effect in PPP approach, biosynthesis and antioxidant Metabolism.
Current existing codehydrogenase Ⅱ NADP+, should or NADPH content assaying methods are 6 glucose phosphate dehydrogenase coupling methods The shortcomings that method, is：1st, animal, plant, microorganism and culture cell sample can not be common to；2nd, test limit is too high, Zhi Nengda To 10-3mol/L；3rd, it is larger by impurity interference, false positive be present；4th, it is not high to detect stability.
The content of the invention
The defects of in order to overcome prior art, the present invention is intended to provide a kind of codehydrogenase Ⅱ NADP+Or NADPH assays examination Agent box and its method, it can effectively determine codehydrogenase Ⅱ NADP+Or NADPH content.
To realize above-mentioned technical purpose and the technique effect, the present invention is achieved through the following technical solutions：
A kind of codehydrogenase Ⅱ NADP+Or NADPH assay kits, including following reagent：
Acidic extraction liquid, 50mL × 1 bottle, 50mL is settled to by concentrated hydrochloric acid and formed, 4 DEG C of preservations；
Alkaline extract solution, 50mL × 1 bottle, NaOH are settled to 50mL and formed, 4 DEG C of preservations；
Reagent one, mL × 1 bottle of liquid 15, it is settled to 15mL after being mixed by HEPES with EDTA and forms, and pH to 8.0 is adjusted with KOH, 4 DEG C of preservations；
Reagent two, pulvis × 1, is made up of G6P, is placed in 5mL reagent bottles, -20 DEG C of preservations；
Reagent three, pulvis × 1, is made up of PMS, is placed in 5mL brown bottles, -20 DEG C of preservations；
Reagent four, pulvis × 1, is made up of MTT, is placed in 5mL brown bottles, 4 DEG C of preservations；
Reagent five, liquid 1.8mL × 1, by 14U/mL glucose-6-phosphate dehydrogenase solution composition, it is placed in 2mL EP pipes In, 4 DEG C of preservations；
Reagent six, liquid 30mL × 1 bottle, 30mL is settled to by NaCl and formed, 4 DEG C of preservations；
Reagent seven, liquid 50mL × 1 bottle, is mixed by ethanol and distilled water, 4 DEG C of preservations.
Further, the volume of concentrated hydrochloric acid is 428 μ L in the acidic extraction liquid.
Further, NaOH quality is 0.2g in the alkaline extract solution.
Further, HEPES quality is 0.715g in the reagent one, and EDTA quality is 4.47mg.
Further, the quality of G6P is 30.4mg in the reagent two.
Further, PMS quality is 12.47mg in the reagent three.
Further, MTT quality is 4.26mg in the reagent four.
Further, 14U/mL glucose-6-phosphate dehydrogenase solution is by 25 μ L 6- phosphoric acid in the reagent five Glucose dehydrogenase is dissolved in 1.8mL, is formulated after 50mM, pH7.6 Tris-HCl cushioning liquid.
Further, NaCl quality is 7g in the reagent six.
Further, the volume of ethanol is 48mL in the reagent seven, and the volume of distilled water is 2mL.
A kind of codehydrogenase Ⅱ NADP using mentioned reagent box+Or NADPH content assaying methods, based on AAS, divide Not Yong acidic extraction liquid and alkaline extract solution extraction sample in NADP+And NADPH, NADPH are acted on by PMS hydrogen of passing, and make oxygen Change type tetrazolium bromide（MTT）First a ceremonial jade-ladle, used in libation is reduced to, light absorption value is detected under 570nm, so as to determine NADPH contents, then utilizes 6- phosphorus Sour grapes glucocorticoid dehydrogenase reduces NADP+For NADPH, so as to detect NADP+Content；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare visible spectrophotometer, desk centrifuge, adjustable pipette, 1 mL glass cuvettes, mortar, ice and distillation Water；
The preparation of step 2 reagent before use；
1）4mL distilled water is added in the reagent two, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
2）4mL distilled water is added in the reagent three, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
3）4mL distilled water is added in the reagent four, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
Step 3 NADP+With NADPH extraction；
1）Serum（Slurry）Middle NADP+It is as follows with NADPH extraction, concrete operations：
NADP+Extraction：According to serum（Slurry）Volume（mL）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio（It is recommended that take About 0.1mL serum（Slurry）, add 1mL acidic extraction liquid）, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；It is cold in ice bath But after, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；Take 500 μ L of supernatant liquid, add during 500 μ L alkalescence extract solutions are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
NADPH extraction：According to serum（Slurry）Volume（mL）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio（It is recommended that take About 0.1mL serum（Slurry）, add 1mL alkalescence extract solutions）, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；It is cold in ice bath But after, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；Take 500 μ L of supernatant liquid, add during 500 μ L acidic extraction liquid are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
2）NADP in tissue+With NADPH extraction：
NADP+Extraction：According to liver mass（g）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio（It is recommended that take about 0.1g Tissue, add 1mL acidic extraction liquid）, ice bath grinding, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；Cooled down in ice bath Afterwards, using eccentricity 10000g, at 4 DEG C, 10min is centrifuged；Take 500 μ L of supernatant liquid, add during 500 μ L alkalescence extract solutions are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
NADPH extraction：According to liver mass（g）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio（It is recommended that take about 0.1g Tissue, add 1mL alkalescence extract solutions）, ice bath grinding, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；Cooled down in ice bath Afterwards, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, 500 μ L acidic extraction liquid is added and is allowed to neutralize, Mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
3）NADP in cell or bacterium+With NADPH extraction：
NADP+Extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）： Acidic extraction liquid accumulates（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL acidic extraction liquid）, surpass Sonication（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）, 95 DEG C of water-bath 5min（Cover tightly, to prevent Only moisture loss）；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, are added 500 μ L alkalescence extract solutions are allowed to neutralize, and mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put and treat on ice Survey；
NADPH extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）： Alkaline extracting liquid volume（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL alkalescence extract solutions）, surpass Sonication（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）, 95 DEG C of water-bath 5min（Cover tightly, to prevent Only moisture loss）；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, are added 500 μ L acidic extraction liquid are allowed to neutralize, and mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put and treat on ice Survey；
Step 4 is it will be seen that spectrophotometer preheating more than 30min, adjusting wavelength to 570nm, distilled water zeroing；
Step 5 takes two 1.5mL browns EP pipes, and one is labeled as control tube, and another, labeled as measure pipe, is compareing respectively According to the form below is loaded successively in pipe and measure pipe：
Control tube and measure pipe are fully mixed respectively, after standing 5min, using eccentricity 20000g, 5min is centrifuged at 25 DEG C, Supernatant is abandoned, is added in precipitation：
Control tube and measure pipe are mixed respectively, and utilize visible spectrophotometer colorimetric under 570nm wavelength, reads control tube Light absorption value A1 and measure pipe light absorption value A2, calculates △ A=A2-A1；
Step 6 NADP+With the calculating of NADPH contents；
1）NADP+The calculating of content；
1.1 serum（Slurry）Middle NADP+The calculating of content, its specific calculation are as follows：
NADP+Content（nmol/mL）=[4.57×（△A - 0.062）×V1）]÷（V3×V1÷V2）= 91.4×（△A - 0.062）；
NADP in 1.2 tissues, bacterium or cell+Cubage, its specific calculation are as follows：
A. calculated by sample protein concentration；
NADP+Content（nmol/mg prot）=[4.57×（△A - 0.062）×V1）]÷（V1×Cpr）= 4.57×(△A - 0.062）÷Cpr；
B. calculated by sample fresh weight；
NADP+Content（Nmol/g fresh weights）= [ 4.57×（△A - 0.062）×V1）]÷（W×V1÷V2）= 9.14× （△A - 0.062）÷W；
C. calculated by bacterium or cell density；
NADP+Content（nmol/104 cell）=[ 4.57×（△A - 0.062）×V1）]÷（500×V1÷V2）= 0.0183×（△A - 0.062）；
2）The calculating of NADPH contents；
2.1 serum（Slurry）Middle NADPH cubages, its specific calculation are as follows：
NADPH contents（nmol/mL）=[7.2×（△A - 0.072）×V1）]÷（V3×V1÷V2）= 144×（△A - 0.072）；
NADPH cubages in 2.2 tissues, bacterium or cell, its specific calculation are as follows：
A. calculated by sample protein concentration；
NADPH contents（nmol/mg prot）= [7.2×（△A - 0.072）×V1）]÷（V1×Cpr）= 7.2×（△A - 0.072）÷Cpr；
B. calculated by sample fresh weight；
NADPH contents（Nmol/g fresh weights）= [7.2×（△A - 0.072）×V1） ]÷（W×V1÷V2)=14.4×（△ A - 0.072）÷W；
C. calculated by bacterium or cell density；
NADPH contents（nmol/104 cell）= [7.2×（△A - 0.072）×V1）]÷（500×V1÷V2）=0.0288 ×（△A - 0.072）；
Wherein, V1 represents to add the volume of sample in reaction system, and V1=0.05mL；
V2 represents to add the volume of extract solution, and V2=2mL；
V3 represents to add serum（Slurry）Volume, and V3=0.1mL；
Cpr represents the concentration of sample protein, unit mg/mL；
W represents the quality of sample, unit g；
500 represent cell or total number of bacteria, represent 5,000,000.
The beneficial effects of the invention are as follows：
1st, such kit is there is no on the market at present, kit of the invention has filled up the blank of prior art well, can For effectively determining codehydrogenase Ⅱ NADP+Or NADPH content.
2nd, assay method of the invention extracts NADP in sample with acid and alkaline extract solution respectively+And NADPH, extracted Journey selectivity is higher, eliminates the influence of impurity.
3rd, in assay method method of the invention, NADPH is acted on by PMS hydrogen of passing, and makes oxidized form tetrazolium bromide（MTT）Also Originally it was first a ceremonial jade-ladle, used in libation, light absorption value is detected under 570nm, and so as to determine NADPH contents, recycled glucose-6-phosphate dehydrogenase reduction NADP+For NADPH, so as to detect NADP+Content, acted on by this Cascaded amplification, minimum detection limit can reach 10-6mol/ L。
4th, the assay method scope of application of the invention is wider, and it is each to be applicable to animal, plant, microorganism and culture cell etc. Kind sample.
5th, assay method stability of the invention is high, and the coefficient of variation (CV values) is within 2%.
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 specification, described in detail below with presently preferred embodiments of the present invention.The specific reality of the present invention Mode is applied to be shown in detail by following examples.
Embodiment
Below in conjunction with embodiment, to describe the present invention in detail.
A kind of codehydrogenase Ⅱ NADP+Or NADPH assay kits, including following reagent：
Acidic extraction liquid, 50mL × 1 bottle, 50mL is settled to by concentrated hydrochloric acid and formed, 4 DEG C of preservations；
Alkaline extract solution, 50mL × 1 bottle, NaOH are settled to 50mL and formed, 4 DEG C of preservations；
Reagent one, mL × 1 bottle of liquid 15, it is settled to 15mL after being mixed by HEPES with EDTA and forms, and pH to 8.0 is adjusted with KOH, 4 DEG C of preservations；
Reagent two, pulvis × 1, is made up of G6P, is placed in 5mL reagent bottles, -20 DEG C of preservations；
Reagent three, pulvis × 1, is made up of PMS, is placed in 5mL brown bottles, -20 DEG C of preservations；
Reagent four, pulvis × 1, is made up of MTT, is placed in 5mL brown bottles, 4 DEG C of preservations；
Reagent five, liquid 1.8mL × 1, by 14U/mL glucose-6-phosphate dehydrogenase solution composition, it is placed in 2mL EP pipes In, 4 DEG C of preservations；
Reagent six, liquid 30mL × 1 bottle, 30mL is settled to by NaCl and formed, 4 DEG C of preservations；
Reagent seven, liquid 50mL × 1 bottle, is mixed by ethanol and distilled water, 4 DEG C of preservations.
Further, the volume of concentrated hydrochloric acid is 428 μ L in the acidic extraction liquid.
Further, NaOH quality is 0.2g in the alkaline extract solution.
Further, HEPES quality is 0.715g in the reagent one, and EDTA quality is 4.47mg.
Further, the quality of G6P is 30.4mg in the reagent two.
Further, PMS quality is 12.47mg in the reagent three.
Further, MTT quality is 4.26mg in the reagent four.
Further, 14U/mL glucose-6-phosphate dehydrogenase solution is by 25 μ L 6- phosphoric acid in the reagent five Glucose dehydrogenase is dissolved in 1.8mL, is formulated after 50mM, pH7.6 Tris-HCl cushioning liquid.
Further, NaCl quality is 7g in the reagent six.
Further, the volume of ethanol is 48mL in the reagent seven, and the volume of distilled water is 2mL.
A kind of codehydrogenase Ⅱ NADP using mentioned reagent box+Or NADPH content assaying methods, based on AAS, divide Not Yong acidic extraction liquid and alkaline extract solution extraction sample in NADP+And NADPH, NADPH are acted on by PMS hydrogen of passing, and make oxygen Change type tetrazolium bromide（MTT）First a ceremonial jade-ladle, used in libation is reduced to, light absorption value is detected under 570nm, so as to determine NADPH contents, then utilizes 6- phosphorus Sour grapes glucocorticoid dehydrogenase reduces NADP+For NADPH, so as to detect NADP+Content；
This method comprises the following steps that：
The preparation of step 1 instrument and articles for use；
Prepare visible spectrophotometer, desk centrifuge, adjustable pipette, 1 mL glass cuvettes, mortar, ice and distillation Water；
The preparation of step 2 reagent before use；
1）4mL distilled water is added in the reagent two, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
2）4mL distilled water is added in the reagent three, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
3）4mL distilled water is added in the reagent four, it is standby after mixing；Exhaustless 4 DEG C of reagent preserves one week；
Step 3 NADP+With NADPH extraction；
1）Serum（Slurry）Middle NADP+It is as follows with NADPH extraction, concrete operations：
NADP+Extraction：According to serum（Slurry）Volume（mL）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio（It is recommended that take About 0.1mL serum（Slurry）, add 1mL acidic extraction liquid）, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；It is cold in ice bath But after, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；Take 500 μ L of supernatant liquid, add during 500 μ L alkalescence extract solutions are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
NADPH extraction：According to serum（Slurry）Volume（mL）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio（It is recommended that take About 0.1mL serum（Slurry）, add 1mL alkalescence extract solutions）, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；It is cold in ice bath But after, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；Take 500 μ L of supernatant liquid, add during 500 μ L acidic extraction liquid are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
2）NADP in tissue+With NADPH extraction：
NADP+Extraction：According to liver mass（g）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio（It is recommended that take about 0.1g Tissue, add 1mL acidic extraction liquid）, ice bath grinding, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；Cooled down in ice bath Afterwards, using eccentricity 10000g, at 4 DEG C, 10min is centrifuged；Take 500 μ L of supernatant liquid, add during 500 μ L alkalescence extract solutions are allowed to With, mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
NADPH extraction：According to liver mass（g）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio（It is recommended that take about 0.1g Tissue, add 1mL alkalescence extract solutions）, ice bath grinding, 95 DEG C of water-bath 5min（Cover tightly, to prevent moisture loss）；Cooled down in ice bath Afterwards, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, 500 μ L acidic extraction liquid is added and is allowed to neutralize, Mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
3）NADP in cell or bacterium+With NADPH extraction：
NADP+Extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）： Acidic extraction liquid accumulates（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL acidic extraction liquid）, surpass Sonication（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）, 95 DEG C of water-bath 5min（Cover tightly, to prevent Only moisture loss）；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, are added 500 μ L alkalescence extract solutions are allowed to neutralize, and mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put and treat on ice Survey；
NADPH extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）： Alkaline extracting liquid volume（mL）For 500 ~ 1000：1 ratio（It is recommended that 5,000,000 bacteriums or cell add 1mL alkalescence extract solutions）, surpass Sonication（Ice bath, power 20% or 200W, ultrasonic 3s, 10s is spaced, repeated 30 times）, 95 DEG C of water-bath 5min（Cover tightly, to prevent Only moisture loss）；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, are added 500 μ L acidic extraction liquid are allowed to neutralize, and mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put and treat on ice Survey；
Step 4 is it will be seen that spectrophotometer preheating more than 30min, adjusting wavelength to 570nm, distilled water zeroing；
Step 5 takes two 1.5mL browns EP pipes, and one is labeled as control tube, and another, labeled as measure pipe, is compareing respectively According to the form below is loaded successively in pipe and measure pipe：
Control tube and measure pipe are fully mixed respectively, after standing 5min, using eccentricity 20000g, 5min is centrifuged at 25 DEG C, Supernatant is abandoned, is added in precipitation：
Control tube and measure pipe are mixed respectively, and utilize visible spectrophotometer colorimetric under 570nm wavelength, reads control tube Light absorption value A1 and measure pipe light absorption value A2, calculates △ A=A2-A1；
Step 6 NADP+With the calculating of NADPH contents；
1）NADP+The calculating of content；
1.1 serum（Slurry）Middle NADP+The calculating of content, its specific calculation are as follows：
NADP+Content（nmol/mL）=[4.57×（△A - 0.062）×V1）]÷（V3×V1÷V2）= 91.4×（△A - 0.062）；
NADP in 1.2 tissues, bacterium or cell+Cubage, its specific calculation are as follows：
A. calculated by sample protein concentration；
NADP+Content（nmol/mg prot）=[4.57×（△A - 0.062）×V1）]÷（V1×Cpr）= 4.57×(△A - 0.062）÷Cpr；
B. calculated by sample fresh weight；
NADP+Content（Nmol/g fresh weights）= [ 4.57×（△A - 0.062）×V1）]÷（W×V1÷V2）= 9.14× （△A - 0.062）÷W；
C. calculated by bacterium or cell density；
NADP+Content（nmol/104 cell）=[ 4.57×（△A - 0.062）×V1）]÷（500×V1÷V2）= 0.0183×（△A - 0.062）；
2）The calculating of NADPH contents；
2.1 serum（Slurry）Middle NADPH cubages, its specific calculation are as follows：
NADPH contents（nmol/mL）=[7.2×（△A - 0.072）×V1）]÷（V3×V1÷V2）= 144×（△A - 0.072）；
NADPH cubages in 2.2 tissues, bacterium or cell, its specific calculation are as follows：
A. calculated by sample protein concentration；
NADPH contents（nmol/mg prot）= [7.2×（△A - 0.072）×V1）]÷（V1×Cpr）= 7.2×（△A - 0.072）÷Cpr；
B. calculated by sample fresh weight；
NADPH contents（Nmol/g fresh weights）= [7.2×（△A - 0.072）×V1） ]÷（W×V1÷V2)=14.4×（△ A - 0.072）÷W；
C. calculated by bacterium or cell density；
NADPH contents（nmol/104 cell）= [7.2×（△A - 0.072）×V1）]÷（500×V1÷V2）=0.0288 ×（△A - 0.072）；
Wherein, V1 represents to add the volume of sample in reaction system, and V1=0.05mL；
V2 represents to add the volume of extract solution, and V2=2mL；
V3 represents to add serum（Slurry）Volume, and V3=0.1mL；
Cpr represents the concentration of sample protein, unit mg/mL；
W represents the quality of sample, unit g；
500 represent cell or total number of bacteria, represent 5,000,000.
Above-described embodiment is in the art the purpose is to be to allow simply to illustrate that the technical concepts and features of the present invention Those of ordinary skill can understand present disclosure and implement according to this, and it is not intended to limit the scope of the present invention.It is all It is the equivalent change or modification according to made by the essence of present invention, should all covers within the scope of the present invention.

Claims (10)

  1. A kind of 1. codehydrogenase Ⅱ NADP+Or NADPH assay kits, it is characterised in that including following reagent：
    Acidic extraction liquid, 50mL × 1 bottle, 50mL is settled to by concentrated hydrochloric acid and formed, 4 DEG C of preservations；
    Alkaline extract solution, 50mL × 1 bottle, NaOH are settled to 50mL and formed, 4 DEG C of preservations；
    Reagent one, mL × 1 bottle of liquid 15, it is settled to 15mL after being mixed by HEPES with EDTA and forms, and pH to 8.0 is adjusted with KOH, 4 DEG C of preservations；
    Reagent two, pulvis × 1, is made up of G6P, is placed in 5mL reagent bottles, -20 DEG C of preservations；
    Reagent three, pulvis × 1, is made up of PMS, is placed in 5mL brown bottles, -20 DEG C of preservations；
    Reagent four, pulvis × 1, is made up of MTT, is placed in 5mL brown bottles, 4 DEG C of preservations；
    Reagent five, liquid 1.8mL × 1, by 14U/mL glucose-6-phosphate dehydrogenase solution composition, it is placed in 2mL EP pipes In, 4 DEG C of preservations；
    Reagent six, liquid 30mL × 1 bottle, 30mL is settled to by NaCl and formed, 4 DEG C of preservations；
    Reagent seven, liquid 50mL × 1 bottle, is mixed by ethanol and distilled water, 4 DEG C of preservations.
  2. 2. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The acidity The volume of concentrated hydrochloric acid is 428 μ L in extract solution, and NaOH quality is 0.2g in the alkaline extract solution.
  3. 3. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent HEPES quality is 0.715g in one, and EDTA quality is 4.47mg.
  4. 4. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent The quality of G6P is 30.4mg in two.
  5. 5. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent PMS quality is 12.47mg in three.
  6. 6. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent MTT quality is 4.26mg in four.
  7. 7. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent 14U/mL glucose-6-phosphate dehydrogenase solution is to be dissolved in 1.8mL by 25 μ L glucose-6-phosphate dehydrogenase in five, It is formulated after 50mM, pH7.6 Tris-HCl cushioning liquid.
  8. 8. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent NaCl quality is 7g in six.
  9. 9. codehydrogenase Ⅱ NADP according to claim 1+Or NADPH assay kits, it is characterised in that：The reagent The volume of ethanol is 48mL in seven, and the volume of distilled water is 2mL.
  10. A kind of 10. codehydrogenase Ⅱ NADP using kit as claimed in claim 1+Or NADPH content assaying methods, its feature It is, based on AAS, comprises the following steps that：
    The preparation of step 1 instrument and articles for use；
    Prepare visible spectrophotometer, desk centrifuge, adjustable pipette, 1 mL glass cuvettes, mortar, ice and distillation Water；
    The preparation of step 2 reagent before use；
    1）4mL distilled water is added in the reagent two, it is standby after mixing；
    2）4mL distilled water is added in the reagent three, it is standby after mixing；
    3）4mL distilled water is added in the reagent four, it is standby after mixing；
    Step 3 NADP+With NADPH extraction；
    1）NADP in serum or blood plasma+It is as follows with NADPH extraction, concrete operations：
    NADP+Extraction：According to serum or Plasma volumes（mL）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio, 95 DEG C of water Bathe 5min；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, add 500 μ L alkali Property extract solution be allowed to neutralize, mix, using eccentricity 10000g, 10min is centrifuged at 4 DEG C, takes supernatant, is put to be measured on ice；
    NADPH extraction：According to serum or Plasma volumes（mL）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio, 95 DEG C Water-bath 5min；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, add 500 μ L Acidic extraction liquid is allowed to neutralize, and mixes, using eccentricity 10000g, centrifuges 10min at 4 DEG C, take supernatant, put to be measured on ice；
    2）NADP in tissue+With NADPH extraction：
    NADP+Extraction：According to liver mass（g）：Acidic extraction liquid accumulates（mL）For 1：5 ~ 10 ratio, ice bath grinding, 95 DEG C Water-bath 5min；After being cooled down in ice bath, using eccentricity 10000g, at 4 DEG C, 10min is centrifuged；500 μ L of supernatant liquid are taken, add 500 μ L alkalescence extract solutions are allowed to neutralize, and mix, using eccentricity 10000g, centrifuge 10min at 4 DEG C, take supernatant, put to be measured on ice；
    NADPH extraction：According to liver mass（g）：Alkaline extracting liquid volume（mL）For 1：5 ~ 10 ratio, ice bath grinding, 95 DEG C water-bath 5min；After being cooled down in ice bath, using eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, add 500 μ L acidic extraction liquid is allowed to neutralize, and mixes, using eccentricity 10000g, centrifuges 10min at 4 DEG C, take supernatant, put to be measured on ice；
    3）NADP in cell or bacterium+With NADPH extraction：
    NADP+Extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）：Acid Property extracting liquid volume（mL）For 500 ~ 1000：1 ratio, ultrasonic disruption, 95 DEG C of water-bath 5min；After being cooled down in ice bath, use Eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, 500 μ L alkalescence extract solutions is added and is allowed to neutralize, mix, adopt With eccentricity 10000g, 10min is centrifuged at 4 DEG C, supernatant is taken, puts to be measured on ice；The method of the ultrasonic disruption is first ice bath, Then power 20% or 200W are used, ultrasonic 3s, is spaced 10s, is repeated 30 times；
    NADPH extraction：Cell or bacterium are first collected in centrifuge tube, supernatant is abandoned, according to bacterium or cell quantity（104It is individual）：Alkali Property extracting liquid volume（mL）For 500 ~ 1000：1 ratio, ultrasonic disruption, 95 DEG C of water-bath 5min；After being cooled down in ice bath, use Eccentricity 10000g, 10min is centrifuged at 4 DEG C；500 μ L of supernatant liquid are taken, 500 μ L acidic extraction liquid is added and is allowed to neutralize, mix, adopt With eccentricity 10000g, 10min is centrifuged at 4 DEG C, supernatant is taken, puts to be measured on ice；
    Step 4 is it will be seen that spectrophotometer preheating more than 30min, adjusting wavelength to 570nm, distilled water zeroing；
    Step 5 takes two 1.5mL browns EP pipes, and one is labeled as control tube, and another labeled as measure pipe；
    Sequentially added in control tube 50 μ L samples, 250 μ L reagents one, 75 μ L reagents two, 75 μ L reagents three, 75 μ L reagents four, 35 μ L reagents five and 500 μ L reagents six；
    50 μ L samples, 250 μ L reagents one, 75 μ L reagents two, 75 μ L reagents three, 75 μ L reagents are sequentially added in pipe is determined simultaneously 4th, 35 μ L reagents five, after mixing, after room temperature lucifuge stands 20min, 500 μ L reagents six are added；
    Then control tube and measure pipe are fully mixed respectively, after standing 5min, using eccentricity 20000g, at 25 DEG C from Heart 5min, supernatant is abandoned, add 1000 μ L reagents seven in control tube and the precipitation of measure pipe respectively；
    Finally control tube and measure pipe are mixed respectively, colorimetric, reading control under 570nm wavelength using visible spectrophotometer Pipe light absorption value A1 and measure pipe light absorption value A2, calculates △ A=A2-A1；
    Step 6 NADP+With the calculating of NADPH contents；
    1）NADP+The calculating of content；
    1.1 serum（Slurry）Middle NADP+The calculating of content, its specific calculation are as follows：
    NADP+Content（nmol/mL）=[4.57×（△A - 0.062）×V1）]÷（V3×V1÷V2）= 91.4×（△A - 0.062）；
    NADP in 1.2 tissues, bacterium or cell+Cubage, its specific calculation are as follows：
    A. calculated by sample protein concentration；
    NADP+Content（nmol/mg prot）=[4.57×（△A - 0.062）×V1）]÷（V1×Cpr）= 4.57×(△A - 0.062）÷Cpr；
    B. calculated by sample fresh weight；
    NADP+Content（Nmol/g fresh weights）= [ 4.57×（△A - 0.062）×V1）]÷（W×V1÷V2）= 9.14× （△A - 0.062）÷W；
    C. calculated by bacterium or cell density；
    NADP+Content（nmol/104 cell）=[ 4.57×（△A - 0.062）×V1）]÷（500×V1÷V2）= 0.0183×（△A - 0.062）；
    2）The calculating of NADPH contents；
    2.1 serum（Slurry）Middle NADPH cubages, its specific calculation are as follows：
    NADPH contents（nmol/mL）=[7.2×（△A - 0.072）×V1）]÷（V3×V1÷V2）= 144×（△A - 0.072）；
    NADPH cubages in 2.2 tissues, bacterium or cell, its specific calculation are as follows：
    A. calculated by sample protein concentration；
    NADPH contents（nmol/mg prot）= [7.2×（△A - 0.072）×V1）]÷（V1×Cpr）= 7.2×（△A - 0.072）÷Cpr；
    B. calculated by sample fresh weight；
    NADPH contents（Nmol/g fresh weights）= [7.2×（△A - 0.072）×V1） ]÷（W×V1÷V2)=14.4×（△ A - 0.072）÷W；
    C. calculated by bacterium or cell density；
    NADPH contents（nmol/104 cell）= [7.2×（△A - 0.072）×V1）]÷（500×V1÷V2）=0.0288 ×（△A - 0.072）；
    Wherein, V1 represents to add the volume of sample in reaction system, and V1=0.05mL；
    V2 represents to add the volume of extract solution, and V2=2mL；
    V3 represents to add serum（Slurry）Volume, and V3=0.1mL；
    Cpr represents the concentration of sample protein, unit mg/mL；
    W represents the quality of sample, unit g；
    500 represent cell or total number of bacteria, represent 5,000,000.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109060791A (en) * 2018-09-06 2018-12-21 苏州科铭生物技术有限公司 A kind of glutaminase active assay kit and its method based on micromethod
CN109609594A (en) * 2018-12-24 2019-04-12 苏州科铭生物技术有限公司 A kind of glucose 6-phosphate assay kit and method based on micromethod
CN115078341A (en) * 2022-08-22 2022-09-20 上海执诚生物科技有限公司 Reagent for detecting glucose-6-phosphate dehydrogenase and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11194120A (en) * 1997-08-28 1999-07-21 Nippon Kasei Chem Co Ltd Method and apparatus for quantitative analysis of mixed acid solution in etching process as well as etching control method and preparation of the mixed acid solution
CN1366550A (en) * 2000-02-16 2002-08-28 大赛璐化学工业株式会社 (R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same
JP2004065078A (en) * 2002-08-05 2004-03-04 Niigata Prefecture Kit for simply determining concentration of pyruvic acid and method for simply determining concentration of pyruvic acid
CN101419172A (en) * 2008-09-25 2009-04-29 上海锦隆生物科技有限公司 A kind of ethanol content detecting reagent in saliva
CN102061285A (en) * 2010-11-26 2011-05-18 上海交通大学 Application method for medicament based on NAD<+> and NADH
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)

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH11194120A (en) * 1997-08-28 1999-07-21 Nippon Kasei Chem Co Ltd Method and apparatus for quantitative analysis of mixed acid solution in etching process as well as etching control method and preparation of the mixed acid solution
CN1366550A (en) * 2000-02-16 2002-08-28 大赛璐化学工业株式会社 (R)-2-octanol dehydrogenase, process for producing enzyme, DNA encoding enzyme and process for producing alcohol by using same
JP2004065078A (en) * 2002-08-05 2004-03-04 Niigata Prefecture Kit for simply determining concentration of pyruvic acid and method for simply determining concentration of pyruvic acid
CN101419172A (en) * 2008-09-25 2009-04-29 上海锦隆生物科技有限公司 A kind of ethanol content detecting reagent in saliva
CN102061285A (en) * 2010-11-26 2011-05-18 上海交通大学 Application method for medicament based on NAD<+> and NADH
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 (1)

* Cited by examiner, † Cited by third party
Title
陈蓉: "《人体氧化还原态与中医阴阳平衡的关系初探》", 《中国优秀硕士论文全文数据库》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109060791A (en) * 2018-09-06 2018-12-21 苏州科铭生物技术有限公司 A kind of glutaminase active assay kit and its method based on micromethod
CN109609594A (en) * 2018-12-24 2019-04-12 苏州科铭生物技术有限公司 A kind of glucose 6-phosphate assay kit and method based on micromethod
CN115078341A (en) * 2022-08-22 2022-09-20 上海执诚生物科技有限公司 Reagent for detecting glucose-6-phosphate dehydrogenase and application thereof
CN115078341B (en) * 2022-08-22 2022-11-29 上海执诚生物科技有限公司 Reagent for detecting glucose-6-phosphate dehydrogenase and application thereof

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