CN107505379A - A kind of method of capillary electrophoresis separation wheat gliadin - Google Patents
A kind of method of capillary electrophoresis separation wheat gliadin Download PDFInfo
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Abstract
The invention discloses a kind of method of capillary electrophoresis separation wheat gliadin.Methods described comprises the following steps：Using cationic polyvinylpyrrolidone as material, the prepares coating on the tube wall of capillary；Using capillary electrophoresis separation wheat samples, that is, realize the separation of wheat gliadin；The cationic polyvinylpyrrolidone is the polyvinylpyrrolidone of cationization, and viscosity average molecular weigh is 20000~25000；The coating is prepared as steps described below：The quartz capillary is rinsed using the aqueous solution and running buffer A of the cationic polyvinylpyrrolidone successively.The present invention is used as coating material using the polyvinylpyrrolidone (CPVP) being cationized, dynamic embellishment is carried out to capillary column, pass through optimization, filter out the suitable extracting separation condition of wheat gliadin, establish a stable separation wheat alcohol soluble protein technical method, the wheat gliadin of different cultivars can be effectively distinguished, turns into a kind of wheat breed finger-print isolation technics.
Description
Technical field
The present invention relates to a kind of method of capillary electrophoresis separation wheat gliadin.
Background technology
Alcohol soluble protein is the main storage protein in wheat seed endosperm.In its mature seed, alcohol soluble protein accounts for always
The 40~50% of albumen.Alcohol soluble protein is single subunit in structure, and through acid polyacrylamide gel electrophoresis, its electrophoretic band is pressed
Molecular size range is different with mobility, is divided into the type of α, β, γ and ω tetra-.Alcohol soluble protein composition has polymorphism, in wheat product
There is notable difference in inter-species, the banding pattern of its electrophoresis pattern is mainly controlled by genotype, therefore gliadin profiles can be made
It is used for cultivar identification, Seed purity test, Genetic relationship etc. for finger-print.
The separation method of alcohol soluble protein mainly includes acid polyacrylamide gel electrophoresis, reverse phase HPLC chromatogram at present
Method and Capillary Electrophoresis.Wherein acid polyacrylamide gel electrophoresis are the analysis sides announced in 1986 of international Seed Inspection association
Method, it is widely used.This method is disadvantageous in that time-consuming, laborious and resolution ratio is low, and needs to use toxic reagent.
Capillary electrophoresis separation albumen has the characteristics that quick, efficient, high resolution, has for vegetative storage protein separation unique excellent
Gesture.But capillary wall absorption is to separate a problem of alcohol soluble protein, it is therefore desirable to which capillary electrophoresis separation method is carried out
Improve.
The content of the invention
It is an object of the invention to provide a kind of method of capillary electrophoresis separation wheat gliadin, the inventive method is with sun
The polyvinylpyrrolidone (CPVP, cationic polyvinylpyrrolidone) of ionization is used as coating material, with to capillary column
Dynamic embellishment is carried out, is then obtained by Optimization of Wheat Extraction of Zein, running buffer system etc.；The inventive method
The wheat gliadin of different cultivars can be effectively distinguished, for a kind of fast and efficiently wheat breed finger-print separation method.
The method of capillary electrophoresis separation wheat gliadin provided by the present invention, comprises the following steps：
Using cationic polyvinylpyrrolidone as material, the prepares coating on the tube wall of capillary；Using capillary
Wheat samples are separated by electrophoresis, that is, realize the separation of wheat gliadin.
In above-mentioned method, the cationic polyvinylpyrrolidone can be dimethyl diallyl ammonium chloride and ethene
The copolymer of pyrrolidones, its viscosity average molecular weigh can be 20000~25000.
The cationic polyvinylpyrrolidone can specifically be prepared as follows to obtain：
Equipped with magnetic stirring apparatus, thermometer and logical N2A certain amount of dimethyl diallyl chlorine is added in the reactor of pipe
Change ammonium (diallyldimethyl ammonium chlorid DADMAC), vinylpyrrolidone (vinylpyrrolidon
VP) (the two mass ratio is 1：4), initiator 2,2 one azo diisobutyl amidine dihydrochlorides (for the 0.25% of monomer mass), add
Enter a certain amount of distilled water dissolving, it is 0.5g/mL to make VP concentration.65 DEG C are warming up to, 5h is reacted, obtains colloid, add suitable quantity of water to dissolve,
Then acetone precipitation is used.Filter out and precipitate and be dried in vacuo as subject copolymers.
In above-mentioned method, the coating is prepared as steps described below：
The quartz wool is rinsed using the aqueous solution and running buffer A of the cationic polyvinylpyrrolidone successively
Tubule；
The step of aqueous solution of the cationic polyvinylpyrrolidone includes standing after rinsing, such as stand 4~10 points
Clock；
The time that the aqueous solution of the cationic polyvinylpyrrolidone rinses can be 10~20 minutes, pressure is 15~
25psi, such as rinsed 10 minutes in 20psi；
The time that the running buffer A is rinsed can be 10~20 minutes, and pressure is 15~25psi, such as be rinsed in 20psi
10 minutes；
Before sample introduction, in addition to the step of high voltage pre-separation, pre-separation 2 minutes such as under 12kv high voltage.
In above-mentioned method, the mass percent concentration of the aqueous solution of the cationic polyvinylpyrrolidone can be
0.1~0.5%, such as 0.5%；
The running buffer A can be the mixed liquor of the phosphate buffer that pH is 6.5 and acetonitrile；
The concentration of the phosphate buffer is 10mM~20mM；
The volume ratio of the phosphate buffer and the acetonitrile is 4：1.
In above-mentioned method, the condition of the capillary electrophoresis separation is as follows：
Voltage is 12~18kv, preferably 12kv；
Running buffer B is the mixed liquor of the phosphate buffer that pH is 6.5 and acetonitrile；
The concentration of the phosphate buffer is 10mM~20mM, preferably 10mM；
The volume ratio of the phosphate buffer and the acetonitrile is 4：1；
Time can be 25~40 minutes；
Detection wavelength can be 214nm；
Capillary temperature can be 25 DEG C；
Sample tray temperature can be 20 DEG C.
In above-mentioned method, the wheat samples are through following pretreatment：
Wheat seed crushing, after drying and sieving, carry out following extraction step：
1) extracted using sodium-chloride water solution, supernatant A is taken after centrifugation；
2) supernatant A is extracted using sodium-chloride water solution, precipitation is taken after centrifugation；
3) precipitation is extracted using ethanol water, takes supernatant B to be used for the capillary electrophoresis separation after centrifugation.
In above-mentioned method, the baking step can be：Toasted one week under the conditions of 37 DEG C；100 mesh sieves can be crossed.
In above-mentioned method, the concentration of the sodium-chloride water solution can be 0.1~0.2M, concretely 0.1M；
The volumn concentration of ethanol can be 70~75%, concretely 75% in the ethanol water.
In above-mentioned method, in step 1), the time of the extraction can be 15~60 minutes, such as 30 minutes,
The dosage of the sodium-chloride water solution can be：100mg samples add sodium-chloride water solution described in 1mL；
It can be centrifuged under conditions of 12000r/min 10 minutes；
In step 2), the time of the extraction can be 15~60 minutes, such as 30 minutes；
The dosage of the sodium-chloride water solution can be：100mg samples add sodium-chloride water solution described in 1mL；
It can be centrifuged under conditions of 12000r/min 10 minutes；
In step 3), the time of the extraction can be 3~4 hours, such as 3 hours；
It can be centrifuged under conditions of 12000r/min 10 minutes.
Polyvinylpyrrolidone (CPVP) of the present invention to be cationized enters Mobile state as coating material to capillary column
Modification, by optimization, the suitable extracting separation condition of wheat gliadin is filtered out, it is molten to establish a stable separation wheat alcohol
Protein techniques method, the wheat gliadin of different cultivars can be effectively distinguished, turn into a kind of wheat breed finger-print separation skill
Art.
Brief description of the drawings
Fig. 1 is the uv absorption spectra for the CPVP that the present invention uses.
Fig. 2 is the separating resulting using gliadin standard items during various concentrations running buffer.
Fig. 3 is the separating resulting using gliadin standard items during different separation voltages.
Fig. 4 is the separating resulting using gliadin standard items during different pretreatments method.
Fig. 5 is the separation qualification result of 5 kinds of wheat gliadin samples.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The CPVP (cationic polyvinylpyrrolidone) used in following embodiments according to document report, (fawn, Lin Mei by woods
Jade, Zhu Wei etc., the preparation of cationic polyvinylpyrrolidone and its load RNA performances.Applied chemistry, volume 2014,31 the 6th
Phase.), synthesize the copolymer.
Specific method is：Equipped with magnetic stirring apparatus, thermometer and logical N2A certain amount of diformazan is added in the reactor of pipe
Base diallyl ammonium chloride (diallyldimethyl ammonium chlorid, DADMAC), vinylpyrrolidone
(vinylpyrrolidon, VP) (the two mass ratio is 1：4), initiator 2,2- azo diisobutyl amidine dihydrochlorides are (for monomer
Quality 0.25%), add the dissolving of a certain amount of distilled water, it is 0.5g/mL to make VP concentration.65 DEG C are warming up to, 5h is reacted, obtains glue
Body, add suitable quantity of water to dissolve, then use acetone precipitation.Filter out and precipitate and be dried in vacuo as subject copolymers.
The viscous of CPVP is tested：
1M more than sodium chloride solution 500mL is first configured, later all solvents calculate as solvent.Specifically：Weigh
58.5g sodium chloride, water about 970mL is added, last constant volume is 1000mL；C1 is configured with saline solution：0.01g/mL polymer
Solution 50mL (determining concentration) and C2：0.04g/mL.Specifically：CPVP 0.51215g are weighed, are dissolved overnight, most with 1M sodium chloride
Constant volume is 50mL afterwards, and it is C1=0.01024g/mL to obtain concentration；CPVP2.01601g is weighed, is dissolved overnight, most with 1M sodium chloride
Constant volume is 50mL afterwards, and it is C2=0.04032g/mL to obtain concentration；Test in 30 DEG C of thermostatic baths, measured with dark type viscometer
The delivery time of pure sodium chloride solution is designated as t0：The delivery time for measuring polymer solution C1 is t5.C2 solution is diluted and prepared
Go out 0.002；0.004；0.006；0.008g/mL each 50mL of solution.Viscosity tube is cleaned, finally with tested solution rinse on a small quantity
Tested solution is added after viscosity tube 3 times, is tested the delivery time of solution, and corresponding concentration and time.Specifically：Measure C1 solution
10mL, it is 50mL with salt solution constant volume；C1 solution 20mL are measured, are 50mL with salt solution constant volume；C2 solution 7.5mL are measured, use salt solution
Constant volume is 50mL；C2 solution 10mL are measured, are 50mL with salt solution constant volume；
Therefore it is 2.048mg/mL that each sample concentration, which is respectively No. 1,；No. 2 are 4.096mg/mL；No. 3 are 6.048mg/mL；4
Number it is 8.064mg/mL；
The dark type viscometer of table 1 measures the specific viscosity of different sample concentrations and relative log viscosities
Reduced viscosity when being extrapolated to 0 concentration for 0.01592 milliliter/milligram=0.01592 liter/gram=0.1592 point
Rise/gram, according to Mark-Houwink formula [η]=kMα, that is, obtain bonded restorations.Calculate CPVP average molecular matter
K=1.4 × 10 during amount-4, to calculate, can obtain its viscosity average molecular weigh is for α=0.7：(0.1592/1.4×10-4)^(1/
0.7)=2.3 × 104, therefore the relative viscosity average molecular weigh of the polymer is about 23000.
CPVP Ultraviolet Absorption Characteristics measure：
It is the 0.5%CPVP aqueous solution to take quality volume fraction, and it is determined in 200~300nm using ultraviolet specrophotometer
Light absorbs, obtain the ultra-violet absorption spectrum of the material.
Its uv absorption spectra is as shown in Figure 1.It will be seen from figure 1 that CPVP has light absorbs, absorption maximum in ultra-violet (UV) band
Wavelength is located at 218nm, and therefore, CPVP should not be added in running buffer in electrophoresis process.
Embodiment 1, capillary dynamic coating segregation wheat gliadin
First, material
Gliadin standard items, purchased from Tokyo HuaCheng Industry Co., Ltd.
5 wheat breeds are respectively all wheats 22, Jimai 22, good star 99, short anti-58 and Laishou 953.
The preparation of gliadin standard items：50mg gliadin standard items accurately are weighed, are dissolved in 2mL, 75% (body
Product) in ethanol water, final concentration 25mg/mL.
The preparation of wheat seed sample：Wheat seed directly crushes, and after one week is toasted at 37 DEG C, crosses 100 mesh sieve, then
By the following method one and method two extracted：
Method one：100mg wheat seed samples accurately are weighed, are separately added into 1mL0.1M sodium-chloride water solutions, room temperature concussion
Extraction 30 minutes, centrifugation (12000r/min) 10 minutes, abandons supernatant, adds 1mL0.1M sodium-chloride water solutions, room temperature concussion again
Supernatant is abandoned in extraction 30 minutes, centrifugation (12000r/min) 10 minutes, and precipitation is carried with the concussion of 75% (volume) ethanol water room temperature
Take 3 hours, centrifugation (12000r/min) 10 minutes, supernatant is taken, for Capillary Electrophoresis.
Method two：100mg wheat seed samples accurately are weighed, with 75% (volume) ethanol water room temperature concussion extraction 3
Hour, centrifugation (12000r/min) 10 minutes, supernatant is taken, in order to prevent sample from evaporating, seals up paraffin oil (Advanced
Analytical.FS-SMO15), for Capillary Electrophoresis.
2nd, Capillary Electrophoresis
Hebei Yongnian quartz capillary (50.2cm, effective length 40cm, 50 μm of capillary inner diameter).
New pipe activation：1NNaOH 20psi are rinsed 20 minutes, and ultra-pure water 20psi is rinsed 10 minutes, running buffer 20psi
Rinse 20 minutes it is standby.Running buffer is that various concentrations (10mM~50mM) pH6.5 phosphate buffer (contains 20% second
The volume ratio of nitrile, i.e. phosphate buffer and acetonitrile is 4：1).
The preparation of Dynamic coating：Rinsed 10 minutes with 0.5wt% CPVP aqueous solution 20psi, stand 4 minutes, operation is slow
Fliud flushing 20psi is rinsed 10 minutes, high voltage 12kv pre-separations 2 minutes.
Sample introduction：Hydrodynamic injection 0.5psi, 5 seconds.
Capillary electrophoresis separation：Reverse 12~the 25kv of separation voltage, Detection wavelength 214nm, capillary temperature is 25 DEG C, sample
Product temperature of tray is 20 DEG C, electrophoresis time 25~40 minutes.
3rd, separating resulting and analysis
1st, the influence that various concentrations running buffer separates to gliadin standard items
Using various concentrations (10~50mM) running buffer, collection of illustrative plates such as Fig. 2 institutes of separate gliadins standard items
Show.
As seen from Figure 2, the optimal separation concentration of gliadin standard items is 10mM, with running buffer concentration
Rise, separative efficiency be deteriorated.Therefore preferably 10~20mM phosphate buffer is molten as running buffer, separation wheat alcohol
Albumen.
2nd, the influence that different separation voltages separate to gliadin standard items
Running buffer, different separation voltages are used as using 10mM, pH6.5 phosphate buffer (containing 20% acetonitrile)
Under (12kv, 18kv and 25kv), the collection of illustrative plates of separate gliadins standard items is as shown in Figure 3.
As seen from Figure 3, the optimal separation voltage of gliadin standard items is 12kv, with the liter of separation voltage
Height, separative efficiency are deteriorated.Therefore preferably 12kv~18kv separation voltage, more preferably 12kv separation voltage separation wheat alcohol is molten
Albumen.
3rd, comparison of the Different Extraction Method to alcohol soluble protein extraction effect
In order to fully extract alcohol soluble protein, the present invention compares 2 kinds of extracting method separation alcohol soluble proteins.
Method one is extracted in advance using 0.1M sodium chloride, removes the interference of the water-solubility protein such as albumin and globulin, so
Afterwards alcohol soluble protein is extracted with 75% ethanol water.Method two is directly to extract alcohol soluble protein with 75% ethanol water.
As a result it is as shown in Figure 4, it can be seen that short anti-58 samples of wheat breed extract obtained Capillary Electrophoresis through method one
Peak number amount is significantly more than method two, illustrates that 0.1M sodium chloride extracts in advance, removes the dry of the water-solubility protein such as albumin and globulin
Disturb very necessary for later capillary electrophoresis separation identification finger-print.Therefore the inventive method method for optimizing one pre-processes
Wheat samples.
4th, the alcohol soluble protein finger-print of Wheat Cultivars
Using 10mM, pH6.5 phosphate buffer (containing 20% acetonitrile) as running buffer, separate all wheats 22, Jimai 22,
Good star 99, short anti-5 kinds of wheat gliadin samples such as 58 and Laishou 953, as a result as shown in Figure 5.
As seen from Figure 5, there is notable difference in the electrophoresis pattern of Wheat Cultivars, characteristic remarkable, obtain very well
Resolution.
The present invention is extracted in advance using 0.1M sodium-chloride water solutions it can be seen from above-mentioned analysis, then with 75% ethanol water
The extracting method of solution, using the polyvinylpyrrolidone (CPVP) of cationization as coating material, using 12~18kv (preferably
12kv) separation voltage, 10~20mM (preferably 10mM) pH6.5 phosphate buffers are used as runtime buffer at (containing 20% acetonitrile)
Liquid, the separation detection of alcohol soluble protein is carried out to 5 kinds of wheat gliadins, obtained the high separation qualification result of identification.
Polyvinylpyrrolidone (CPVP) of the present invention to be cationized enters Mobile state as coating material to capillary column
Modification, by optimization, the suitable extracting separation condition of wheat gliadin is filtered out, it is molten to establish a stable separation wheat alcohol
Protein techniques method, the wheat gliadin of different cultivars can be effectively distinguished, turn into a kind of wheat breed finger-print separation
Method.
Claims (8)
1. a kind of method of capillary electrophoresis separation wheat gliadin, comprises the following steps：
Using cationic polyvinylpyrrolidone as material, the prepares coating on the tube wall of capillary；Using Capillary Electrophoresis
Separation wheat sample, that is, realize the separation of wheat gliadin.
2. according to the method for claim 1, it is characterised in that：The cationic polyvinylpyrrolidone is dimethyl two
The copolymer of allyl ammonium chloride and vinylpyrrolidone, its viscosity average molecular weigh are 20000~25000.
3. method according to claim 1 or 2, it is characterised in that：The coating is prepared as steps described below：
The quartzy capillary is rinsed using the aqueous solution and running buffer A of the cationic polyvinylpyrrolidone successively
Pipe；
The step of aqueous solution of the cationic polyvinylpyrrolidone includes standing after rinsing.
4. according to the method any one of claim 1-3, it is characterised in that：The cationic polyvinylpyrrolidone
The aqueous solution mass percent concentration be 0.1~0.5%；
The running buffer A is the mixed liquor of the phosphate buffer that pH is 6.5 and acetonitrile；
The concentration of the phosphate buffer is 10mM~20mM；
The volume ratio of the phosphate buffer and the acetonitrile is 4：1.
5. according to the method any one of claim 1-4, it is characterised in that：The condition of the capillary electrophoresis separation is such as
Under：
Voltage is 12~18kv；
Running buffer B is the mixed liquor of the phosphate buffer that pH is 6.5 and acetonitrile；
The concentration of the phosphate buffer is 10mM~20mM；
The volume ratio of the phosphate buffer and the acetonitrile is 4：1；
Time is 25~40 minutes.
6. according to the method any one of claim 1-5, it is characterised in that：The wheat samples are through following pretreatment：
Wheat seed crushing, after drying and sieving, carry out following extraction step：
1) extracted using sodium-chloride water solution, supernatant A is taken after centrifugation；
2) supernatant A is extracted using sodium-chloride water solution, precipitation is taken after centrifugation；
3) precipitation is extracted using ethanol water, takes supernatant B to be used for the capillary electrophoresis separation after centrifugation.
7. according to the method for claim 6, it is characterised in that：The concentration of the sodium-chloride water solution is 0.1~0.2M；
The volumn concentration of ethanol is 70~75%, concretely 75% in the ethanol water.
8. the method according to claim 6 or 7, it is characterised in that：In step 1), the time of the extraction is 15~60 points
Clock；
In step 2), the time of the extraction is 15~60 minutes；
In step 3), the time of the extraction is 3~4 hours.
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| CN108593394A (en) * | 2018-04-18 | 2018-09-28 | 湖北民族学院 | The acid polyacrylamide gel electrophoresis separation method of Magnoliacea plant seeds prolamine |
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