CN104374768B - Colorimetric method for rapid quantitative determination of amino acid decarboxylases - Google Patents
Colorimetric method for rapid quantitative determination of amino acid decarboxylases Download PDFInfo
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Abstract
The invention discloses a colorimetric method for rapid quantitative detection of amino acid decarboxylases, through the configuration of a reaction liquid and a developing liquid, a standard curve is prepared, and enzyme activity of the amino acid decarboxylases of a to-be-detected sample can be obtained. The method has the advantages of being low in cost, good in repeatability, good in linear relationship in the enzyme concentration of 0.001U-0.006U / muL, simple in operation, low in instrument requirements, reliable, wide in applicable range, and capable of detecting a variety of amino acid decarboxylases. The method is very important for change of the enzyme activity of the amino acid decarboxylases in animal tissues, microbial culture fluids, cell culture and blood samples.
Description
Technical field
The present invention relates to Enzyme assay field, and in particular to a kind of colorimetric side of Quantitative detection amino acid decarboxylases
Method.
Background technology
Amino acid decarboxylases (amino acid decarboxylase) are the carboxyls that certain aminoacid is sloughed in catalysis, are generated
Correspondence amine (as it is following it is parenthetic shown in) lyases general name：NH2CHRCOOH→NH2CH2R+CO2.Mainly in acid training
Find in the antibacterial grown on foster base, although in animal, plant seldom, but it is also possible to find.It is known in antibacterial to have
The phases such as lysine (cadaverine), L-Tyrosine (tyramine), arginine (agmatine), ornithine (putrescine), glutamic acid (γ-aminobutyric acid)
The narrow spectrum decarboxylase answered, these enzymes are with pyridoxal 5-phosphate as coenzyme.There is glutamic acid (gamma-amino in animal tissue
Butanoic acid), L-Tyrosine (tyramine), histidine (histamine), cysteine (taurine), 5-hydroxyryptophan (5-hydroxy tryptamine) it is corresponding specially
The decarboxylase of one property, more with pyridoxal 5-phosphate as coenzyme.It is to give birth to that the amine that amino acid decarboxylase is generated has in animal body many
In reason, pharmacologically play an important role, there is the effect of neutralization acid medium in antibacterial.But there is presently no and quickly determine
The detailed method of amount detection amino acid decarboxylase enzyme activity and the reagent test box of commercialization.It is high in order to substitute traditional cost,
The detection methods such as radioactive material contamination (need liquid scintillation instrument), patent report is had at present and is coupled fixed CO using carboxylase enzyme2
Technology for detection amino acid decarboxylase enzyme activity, but operational approach is not detailed enough, operates highly difficult.Based on this principle, this
It is bright to be intended to invent a kind of colorimetric method of Quantitative detection amino acid decarboxylases, the detection kit of commercialization is developed, formed
Operation step feasible in detail is poly-, and this is for the ammonia in research animal tissue, microbial culture medium, cell culture and blood sample
Base acid decarboxylase vigour changes are most important.
The content of the invention
The purpose of the present invention is to there are provided a kind of colorimetric method of Quantitative detection amino acid decarboxylases, and method is easy
OK, it is with low cost, can complete to detect work in 70 minutes, and only need the routine instrument such as thermostat water bath and spectrophotometer
Device.Broad spectrum activity is strong, can simultaneously correspond to detection several amino acids.Effect stability, it is easy to operate.
In order to achieve the above object, the present invention adopts following technical measures：
A kind of colorimetric method of Quantitative detection amino acid decarboxylases, it is comprised the following steps that：
(1) reactant liquor is constituted with nitrite ion reagent and prepared
Reactant liquor：I.e. with using, reagent 2 is dissolved in reagent 1.
Reagent 1：50mmol/L Tris (pH7.5), 10mmol/L MgCl2, the monocyclic amine salt of 2mmol/LPEP, 5mmol/
L aminoacid, using without CO2Distilled water is prepared；
Described aminoacid is the corresponding aminoacid of amino acid decarboxylases to be detected；
Reagent 2：5U/mg PEP carboxylases；
Nitrite ion：Reagent 3：The solid purple B of 50mg/ml, 1% tween 80 solution, using without CO2Distilled water is prepared；
Reagent 4：Decarboxylase standard substance.
Above solution is adopted without CO2Distilled water is prepared, without CO2The manufacture method of distilled water：Distilled water is heated before use
To after seething with excitement, continue to heat 5min, seal after cooling stand-by.
(2) standard curve preparation of reagents
Decarboxylase standard substance are configured to into 0,0.001,0.002,0.003,0.004,0.005,0.006,0.007,
The enzymatic solution of 0.008U/ μ L Concentraton gradient, solvent is homogenate medium.
PH 7.4,0.01mol/L Tris-Hcl, 0.0001mol/L EDTA-2Na, 0.01mol/L sucrose, 0.8%
Sodium chloride solution.
(3) preparation of standard curve
1. 450 μ L reactant liquors are pipetted to centrifuge tube；
2. the standard substance prepared in 50 μ L steps (2) are added, is turned upside down for several times, mixed；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；
4. in adding 25 μ L nitrite ions and reaction system, turn upside down for several times, mix；
5. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe absorbance (OD) value；
6. experimental procedure is repeated 1. to 5. 9 times；
7. standard curve is built：Vertical coordinate (Y-axis) is the actual read number of absorbance unit；Abscissa (X-axis) is standard decarboxylation
Enzyme enzyme activity unit (U), is standard substance enzyme concentration × 50 μ L；
(4) testing sample assay method
1. 450 μ L reactant liquors are pipetted to centrifuge tube；
2. 50 μ L testing samples or 50 μ L homogenate media are added in centrifuge tube；Turn upside down for several times, mix；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；25 μ L nitrite ions are added in reaction system,
Turn upside down for several times, mix；
4. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe light absorption value OD；
5. according to ODIt is actualCorresponding enzyme concentration E of sample is calculated with standard curveIt is actual(U/ μ L), EIt is actualIt is right with the value of curve x
Should be related to for：EIt is actual=x/50.
6. sample Rate activity is calculated：ESample=EIt is actual× diluted sample multiple ÷ sample total protein concentrations.
In testing sample, if the Rate activity of several amino acids decarboxylase in solution to be measured need to be detected, can only with a certain ammonia
Base acid decarboxylase standard curve is defined.
The formula of described homogenate medium is：PH 7.4,0.01mol/L Tris-Hcl, 0.0001mol/L EDTA-
2Na, 0.01mol/L sucrose, 0.8% sodium chloride solution.
In the above scheme, for the preparation of solution to be measured, using the conventional method of this area, preferably take with lower section
Method：
For microbial solution sample, 10000rpm, 5min is centrifuged, incline supernatant, is cleaned repeatedly with normal saline or PBS
Precipitation 2~3 times, then 10000rpm, are centrifuged 5min, confide all supernatant, with normal saline dilution to 0.8OD, take 10mL,
10000rpm, is centrifuged 5min, confides all supernatant, adds 500 μ L homogenate media or normal saline, and centrifuge tube is inserted in ice, with shaking
30 microns of supersound process 20s, are placed in cooled on ice, and repeatedly supersound process 3 times, make cell breakage, put 4 DEG C of desk centrifuges into
15min, speed is 16000g, carefully removes 500 μ L of supernatant liquid and is placed in ice bank to the 1.5mL centrifuge tubes of new pre-cooling, detection
Total protein content is stand-by.
For animal tissue's sample, weigh tissue sample 1g it is stable in 4 DEG C after ice normal saline or PBS repeatedly
Rinsing 2~3 times, must not be less than every time 5mL, eliminate the impurity such as blood, and filter paper is blotted, and is weighed again, in being placed in 10mL centrifuge tubes
It is stand-by in insertion ice.Tissue samples are put in 10mL centrifuge tubes, with liquid-transfering gun take pre-cooling homogenate medium (PH 7.4,
0.01moL/L Tris-Hcl, 0.0001moL/L EDTA-2Na, the sodium chloride solution of 0.01moL/L sucrose 0.8%), homogenate
The volume total amount of medium should be the 9mL of piece of tissue weight, with liquid-transfering gun take the homogenate medium or normal saline of total amount 2/3 in
In centrifuge tube, piece of tissue is shredded in centrifuge tube with the shears of clean pre-cooling, with 10000~15000rpm of tissue refiner
Under grind to form 10% tissue homogenate (homogenate 10s/ time, interval 30s, continuous 3~5 times, whole process needs carry out in frozen water
Until grinding is uniform), finally the medium washes refiner edge of a knife is homogenized by remaining 1/3, liquid is collected in the centrifuge tube of homogenate
In.10% homogenate low temperature low speed centrifuge 2000rpm for preparing or so is centrifuged 10~15min, and careful pipettes supernatant
In being sub-packed in the 1.5mL centrifuge tubes of pre-cooling, detection total protein content is stand-by.
The method of the quick detection cocarboxylase vigor that the present invention is provided, compares compared with method and possesses following characteristics：
1st, it is with low cost；
2nd, there is provided carefully complete laboratory operating procedures；
3rd, reproducible, in enzyme concentration, linear relationship is good between 0.001U~0.006U/ μ L；
4th, simple to operate, instrument requirements are not high；
5th, method reliability, it is widely applicable, can detect several amino acids decarboxylase.
Description of the drawings
Fig. 1 is tyrosine decarboxylase standard curve schematic diagram in embodiment 1.
Specific embodiment
Embodiment 1：
A kind of colorimetric method of Quantitative detection amino acid decarboxylases, it is comprised the following steps that：
By taking hog middle microbial cells sample as an example, faecal flora thalline E.C. 4.1.1.18, glutamic acid decarboxylase are determined
Enzyme, tyrosine decarboxylase, glycine decarboxylase, tryptophan decarboxylase, histidine decarboxylase, methionine decarboxylase and arginine
Decarboxylase.
1. reagent
(1) reactant liquor is constituted with nitrite ion reagent and prepared
The agent prescription of Quantitative detection decarboxylase colorimetric method is shown in Table 1.
Reactant liquor：I.e. with using, reagent 2 is dissolved in reagent 1.
Reagent 1 (100ml)：50mmol/L Tris (pH7.5), 10mmol/L MgCl2, the mono- ring amines of 2mmol/LPEP
Salt, 5mmol/L aminoacid, using without CO2Distilled water is prepared；
Described aminoacid is the corresponding aminoacid of amino acid decarboxylases to be detected；
Reagent 2 (20mg)：5U/mg PEP carboxylases；
Nitrite ion：Reagent 3：The solid tween 80 solution of purple B 1% of 50mg/ml, using without CO2Distilled water is prepared；
Reagent 4 (250mg)：Tyrosine decarboxylase standard substance (USP levels, 0.1U/mg).
Above solution is adopted without CO2Distilled water is prepared, without CO2The manufacture method of distilled water：Distilled water is heated before use
To after seething with excitement, continue to heat 5min, seal after cooling stand-by.
The agent prescription of the colorimetric method of the Quantitative detection amino acid decarboxylases of table 1
AAmino acid classes are determined according to detected decarboxylase species, by molecular weight quality is determined.
BReactant liquor preparation program 1：All reagents in addition to PEP carboxylases are dissolved, adjusts pH value to 7.5, be subsequently adding
PEP carboxylases, are finally settled to 1L, now with the current, and less than 4 DEG C preserve；Reactant liquor preparation program 2：Will be except aminoacid and PEP carboxylics
Change all reagents dissolving outside enzyme and adjust pH value to 7.5, be configured to 4 DEG C of preservations of mother solution, aminoacid and carboxylase are added before measurement,
It is now with the current.
(2) standard curve preparation of reagents
Tyrosine decarboxylase standard substance are configured to into 0,0.001,0.002,0.003,0.004,0.005,0.006,
The enzymatic solution of 0.007,0.008U/ μ L Concentraton gradient, solvent is homogenate medium.
(3) it is homogenized the formula of medium
PH 7.4,0.01mol/L Tris-Hcl, 0.0001mol/L EDTA-2Na, 0.01mol/L sucrose, 0.8%
Sodium chloride solution.
2. experimental technique
(1) microbiological specimens prepare before determining
Collect hog middle Digesta samples, separate microorganism (is under sterile conditions carried out enteric microorganism and chyme point
From separating obtained mixed microorganism is tested), 10000rpm is centrifuged 5min, and incline supernatant, anti-with normal saline or PBS
Multiple cleaning precipitation 2~3 times, then 10000rpm, are centrifuged 5min, confide all supernatant, with normal saline dilution to 0.8OD, take 10mL,
10000rpm, is centrifuged 5min, confides all supernatant, adds 500 μ L homogenate media or normal saline, and centrifuge tube is inserted in ice, with shaking
30 microns of supersound process 20s, are placed in cooled on ice, and repeatedly supersound process 3 times, make cell breakage, put 4 DEG C of desk centrifuges into
15min, speed is 16000g, carefully remove 500 μ L of supernatant liquid be placed in the 1.5mL centrifuge tubes of new pre-cooling it is stand-by in ice bank,
Measure each group faecal flora total protein before experiment to be respectively：30kg pigs：1.64±0.38mg/mL；60kg pigs：0.69±
0.11mg/mL；90kg pigs：0.51±0.36mg/mL.
Below by taking detection tyrosine decarboxylase as an example, its assay method and the range of linearity are illustrated：
(2) standard curve making method
Following operation is carried out according to the reagent of table 2 and sample addition：
1. 450 μ L reactant liquors are pipetted to new 1mL centrifuge tubes；
2. the standard substance prepared in 50 μ L steps 1 (2) are added, is turned upside down for several times, mixed；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；
4. in adding 25 μ L nitrite ions and reaction system, turn upside down for several times, mix；
5. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe absorbance (OD) value；
6. experimental procedure is repeated 1. to 5. 9 times；
7. standard curve is built：Vertical coordinate (Y-axis) is the actual read number of absorbance unit；Abscissa (X-axis) is standard decarboxylation
Enzyme enzyme activity unit (U), is the μ L of standard substance enzyme concentration * 50, and the corresponding OD values of tyrosine decarboxylase standard substance enzyme concentration are shown in
Table 3, standard curve is shown in accompanying drawing 1.
Cocarboxylase unit of activity standard curve is：Y=0.4789x+0.1126, R2=0.998.
8. standard curve is done with different amino acid decarboxylases, standard concentration is respectively 0.0006U/ μ L, 0.0016U/ μ
L, 0.0026U/ μ L, 0.0036U/ μ L, 0.0046U/ μ L, 0.0056U/ μ L, 0.0066U/ μ L (being shown in Table 4).
The reagent of table 2 and sample addition and assay method
The corresponding OD values of the tyrosine decarboxylase standard substance enzyme concentration of table 3
When enzyme concentration is between 0.001U/ μ L to 0.006U/ μ L (not including 0.001U/ μ L and 0.006U/ μ L), linearly
Relation is preferable, therefore should measure result according to sample enzyme concentration and suitably adjust sample concentration；Every time measurement standard curve need to do 3 times with
Upper parallel repetition.
Because experimental principle is by catching CO2Colour developing, therefore the calculating of the enzyme concentration of different types of decarboxylase can be with this
For standard curve.But blank experiment need to be simultaneously done when determining to discharge medicine and color sample error, is entered with absolute OD values
Row is calculated.
The corresponding absolute OD values of the different aminoacids decarboxylase standard substance of table 4
Different cocarboxylase unit of activity standard curves are for (because first concentration and last concentration fall in the range of linearity
Outward, thus cast out statistics)：
Lysine：Y=0.474x+0.1107, R2=0.9998；
Glutamic acid：Y=0.474x+0.1123, R2=0.9992；
L-Tyrosine：Y=0.488x+0.1082, R2=0.9996；
Glycine：Y=0.482x+0.1123, R2=0.9999；
Tryptophan：Y=0.478x+0.1118, R2=0.9999；
Histidine：Y=0.48x+0.1136, R2=0.9999；
Methionine：Y=0.484x+0.1093, R2=0.9992；
Arginine：Y=0.478x+0.112, R2=0.9993.
Result above shows again, because experimental principle is by catching CO2Colour developing, therefore different types of decarboxylase
The calculating of enzyme concentration can be calculated with a kind of this decarboxylase standard curve, and the present invention is bent with the standard of tyrosine decarboxylase
Line is defined and is calculated.
(3) sample determination method (table 2)
1. 450 μ L reactant liquors are pipetted to new 2mL centrifuge tubes；
2. 50 μ L testing samples or 50 μ L homogenate medium (blank) are added in centrifuge tube；Turn upside down for several times, mix；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；In adding 25 μ L nitrite ions and reaction system,
Turn upside down for several times, mix；
4. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe light absorption value OD
5. according to ODIt is actualCorresponding enzyme concentration E of sample is calculated with standard curveIt is actual(U/ μ L), EIt is actualIt is right with the value of curve x
Should be related to for：EIt is actual=x/50.
6. sample Rate activity is calculated：ESample=EIt is actual× diluted sample multiple ÷ sample total protein concentrations
The results are shown in Table 5 as follows：
Hog middle microbial amino acid decarboxylation Rate activity (U/mg prot) of table 5
(4) method validation
A unknown solution to be measured (randomly selecting table 5 with the microbiological specimens criticized) is taken, subpackage 7 is managed, often the μ of pipe 25
L, numbering 1-7.0U/ μ L, 0.002U/ μ L, 0.004U/ μ L, 0.006U μ L/, 0.008U/ μ L are separately added in 1-7 pipes,
The μ L of tyrosine decarboxylase standard solution 25 of 0.010U/ μ L, 0.012U/ μ L, it is even to be measured (right as blank with No. 1 pipe to be fully mixed
According to).Its result such as table 6 (because originally having a tyrosine decarboxylase in sample, therefore No. 7 pipe excessive concentrations are beyond being best suitable for measured value,
Cast out).Standard curve is：Y=0.4452x+0.1143, R2=0.998, measured enzyme concentration value still falls within the mark of structure
In directrix curve scope, there is preferable repeatability.
The corresponding OD values of the tyrosine decarboxylase standard substance enzyme concentration of table 6
Embodiment 2：
Determine pig brain tissue's sample E.C. 4.1.1.18, glutamate decarboxylase, L-Tyrosine by taking pig brain tissue's sample as an example to take off
Carboxylic acid, glycine decarboxylase, tryptophan decarboxylase, histidine decarboxylase and arginine decarboxylase.
1. reagent
(1) reactant liquor is constituted with nitrite ion reagent and prepared
(2) standard curve preparation of reagents
It is in the same manner as in Example 1.
2. experimental technique
(1) microbiological specimens prepare before determining
Weigh respectively 30kg pigs and 90kg pigs cerebral tissue block 1g it is stable in 4 DEG C after ice normal saline or PBS
Rinse repeatedly 2~3 times, 5mL must not be less than every time, eliminate the impurity such as blood, filter paper is blotted, and is weighed again, is placed in 10mL centrifugations
Insert in pipe stand-by in ice.Tissue samples are put in 10mL centrifuge tubes, with liquid-transfering gun take pre-cooling homogenate medium (PH7.4,
0.01moL/L Tris-Hcl, 0.0001moL/L EDTA-2Na, the sodium chloride solution of 0.01moL/L sucrose 0.8%), homogenate
The volume total amount of medium should be 9 times of piece of tissue weight, with liquid-transfering gun take the homogenate medium or normal saline of total amount 2/3 in
In centrifuge tube, piece of tissue is shredded in centrifuge tube with the shears of clean pre-cooling, with 10000~15000rpm of tissue refiner
Under grind to form 10% tissue homogenate (homogenate 10s/ time, interval 30s, continuous 3~5 times, whole process needs carry out in frozen water
Until grinding is uniform), finally the medium washes refiner edge of a knife is homogenized by remaining 1/3, liquid is collected in the centrifuge tube of homogenate
In.10% homogenate low temperature low speed centrifuge 2000rpm for preparing or so is centrifuged 10~15min, and careful pipettes supernatant
It is sub-packed in stand-by in the 1.5mL centrifuge tubes of pre-cooling.With colorimetric method for determining sample total protein content, each group total protein difference is measured
For：30kg pigs：0.93±0.32mg/mL；90kg pigs：0.68±0.26mg/mL.
Below by taking detection tyrosine decarboxylase as an example, its assay method and the range of linearity are illustrated：
(2) assay method
It is same as Example 1.
(3) standard curve making method
It is same as Example 1, it is consistent with batch decarboxylase standard curve of the medicine prepared, therefore standard curve is：
Standard curve is：Y=0.4789x+0.1126, R2=0.998.
(4) sample determination method
It is same as Example 1.
The results are shown in Table 7 as follows：
The pig brain tissue's amino acid decarboxylase enzyme activity (U/mg prot) of table 7
Claims (2)
1. a kind of colorimetric method of Quantitative detection amino acid decarboxylases, it is characterised in that it is comprised the following steps that：
(1) reactant liquor is constituted with nitrite ion reagent and prepared
Reactant liquor：I.e. with using, reagent 2 is dissolved in reagent 1；
Reagent 1：50mmol/L Tris, pH7.5,10mmol/L MgCl2, the monocyclic amine salt of 2mmol/L PEP, 5mmol/L amino
Acid；
Described aminoacid is the corresponding aminoacid of amino acid decarboxylases to be detected；
Reagent 2：5U/mg PEP carboxylases；
Nitrite ion：Reagent 3：The solid purple B of 50mg/ml, 1% tween 80 solution；
Reagent 4：Decarboxylase standard substance
Above solution is adopted without CO2Distilled water is prepared；
(2) standard curve preparation of reagents
Decarboxylase standard substance are configured to into 0,0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008U/ μ L
The enzymatic solution of Concentraton gradient, solvent is homogenate medium；
The formula of described homogenate medium is：PH 7.4,0.01mol/L Tris-Hcl, 0.0001mol/L EDTA-2Na,
0.01mol/L sucrose, 0.8% sodium chloride solution；
(3) preparation of standard curve
1. 450 μ L reactant liquors are pipetted to centrifuge tube；
2. the standard substance prepared in 50 μ L steps (2) are added, is turned upside down for several times, mixed；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；
4. in adding 25 μ L nitrite ions and reaction system, turn upside down for several times, mix；
5. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe absorbance (OD) value；
6. experimental procedure is repeated 1. to 5. 9 times；
7. standard curve is built：Vertical coordinate Y-axis is the actual read number of absorbance unit；Abscissa X-axis is standard decarboxylation enzyme enzyme activity
Unit, the numerical value of unit of activity is standard substance enzyme concentration × 50 μ L；
(4) testing sample assay method
1. 450 μ L reactant liquors are pipetted to centrifuge tube；
2. 50 μ L testing samples or 50 μ L homogenate media are added in centrifuge tube；Turn upside down for several times, mix；
3. 1h is incubated at 37 DEG C, mixing is turned upside down for several times every 10min；Add 25 μ L nitrite ions in reaction system, up and down
Overturn for several times, mix；
4. 5min is stood, 1cm optical paths, distilled water zeroing, 520nm surveys each pipe light absorption value OD；
5. according to ODIt is actualCorresponding enzyme concentration E of sample is calculated with standard curveIt is actual, U/ μ L, EIt is actualWith the corresponding relation of the value of curve x
For：EIt is actual=x/50；
6. sample Rate activity is calculated：ESample=EIt is actual× diluted sample multiple ÷ sample total protein concentrations.
2. colorimetric method according to claim 1, described testing sample, its preparation method is as follows：
For microbial solution sample, 10000rpm, 5min is centrifuged, incline supernatant, and with normal saline or PBS precipitation is cleaned repeatedly
2~3 times, then 10000rpm, 5min is centrifuged, supernatant is confided all, with normal saline dilution to 0.8OD, 10mL, 10000rpm are taken, from
Heart 5min, confides all supernatant, adds 500 μ L homogenate media or 500 μ L normal saline, and centrifuge tube is inserted in ice, micro- with amplitude 30
Rice supersound process 20s, is placed in cooled on ice, and repeatedly supersound process 3 times, make cell breakage, put 4 DEG C of desk centrifuge 15min into,
Speed is 16000g, carefully removes 500 μ L of supernatant liquid and is placed in ice bank to the 1.5mL centrifuge tubes of new pre-cooling, detects total protein
Content is stand-by；
For animal tissue's sample, weigh the ice normal saline after tissue sample 1g stablizes in 4 DEG C or PBS is rinsed repeatedly
2~3 times, 5mL must not be less than every time, eliminate impurity, filter paper is blotted, and is weighed again, be placed in be inserted in 10mL centrifuge tubes and treat in ice
With；Tissue samples are put in 10mL centrifuge tubes, with liquid-transfering gun the homogenate medium of pre-cooling is taken, the volume total amount for being homogenized medium is group
Knit block weight 9 times, the homogenate medium of total amount 2/3 is taken in centrifuge tube with liquid-transfering gun, with the shears of clean pre-cooling by piece of tissue
In shredding centrifuge tube, 10% tissue homogenate is ground to form up and down with 10000~15000rpm of tissue refiner, be homogenized 10s/
It is secondary, interval 30s, continuous 3~5 times, whole process needs to carry out in frozen water up to grinding uniformly, finally even by remaining 1/3
The slurry medium washes refiner edge of a knife, liquid is collected in the centrifuge tube of homogenate；The 10% homogenate low temperature low speed for preparing from
Scheming 2000rpm or so is centrifuged 10~15min, and the careful supernatant that pipettes is sub-packed in the 1.5mL centrifuge tubes of pre-cooling, and detection is total
Protein content is stand-by.
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