CN106124599A - A kind of two-dimensional electrophoresis method for protein and application thereof - Google Patents

A kind of two-dimensional electrophoresis method for protein and application thereof Download PDF

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CN106124599A
CN106124599A CN201610526872.0A CN201610526872A CN106124599A CN 106124599 A CN106124599 A CN 106124599A CN 201610526872 A CN201610526872 A CN 201610526872A CN 106124599 A CN106124599 A CN 106124599A
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protein
electrophoresis
adhesive tape
aquation
isoelectric focusing
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赵君峰
王大红
张彬
孙建瑞
张红梅
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Henan University of Science and Technology
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Henan University of Science and Technology
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q

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Abstract

The invention discloses a kind of two-dimensional electrophoresis method for protein, including first to isoelectric focusing electrophoresis and second to sds page vertical electrophoresis, hydrating condition and deposition condition to focusing electrophoresis are improved respectively, including utilizing hydrating fluid, adhesive tape is carried out aquation, obtain the adhesive tape that aquation is good, and good adhesive tape pH of aquation is 47, wherein containing following components in every 100mL hydrating fluid: the carbamide of 41 42.5g, the thiourea of 15 16g, the CHAPS of 2 2.2g, the DTT of 0.9 1.1g, the IPG Buffer of 2 2.2g, the bromjophenol blue of 0.002 0.003g, and the solvent of hydrating fluid is ddH2O；The voltage of isoelectric focusing electrophoresis and time use and arrange as follows: S1:50v, 10h；S2:250v, 3h；S3:500v, 3h；S4:1000v, 1h；S5:8000v, 3h；S6:8000v, 6 6.5h.During the method application that whole protein is distributed in detecting bacillus amyloliquefaciens stable phase somatic cells, Two-dimensional Gel Electrophoresis resolution is high, reproducible.

Description

A kind of two-dimensional electrophoresis method for protein and application thereof
Technical field
The invention belongs to biological technical field, be specifically related to a kind of two-dimensional electrophoresis method for protein and application thereof.
Background technology
The antibiotic substance that bacillus cereus produces has attracted the attention of numerous experts and scholars.Bacillus cereus antibiotic substance is usual It is divided into two big classes: a class is the antibiotic of Ribosome biogenesis, predominantly Pilus Caprae seu Ovis sulfur class antibiotic (lantibiotics), including Subtilin, subtilosin, ericin etc.；The another kind of lipopeptide antibiotic for non-ribosomal synthesis (lipopeptides) fragrant shepherd's purse plain (fengycin), Surfactin (surfactin), iturin family, are mainly included (iturin, bacillomycin, mycosubtilin), bacilysin etc..Antibacterial lipopeptid is to the multiple gram sun in food Property and negative bacteria all have stronger killing action, can quickly suppress the growth of microorganism, edible after easily by vivo protein enzyme water Solving digestion and have no side effect, activity is very strong in acid condition, has good dissolubility and stability, before being great development The new Type of Preservatives of scape.Fragrant shepherd's purse element, owing to having efficient antifungal activity, has in the field such as food antiseptic, food fresh keeping There is important application prospect, caused and paid close attention to widely.
By the method for Two-Dimensional Gel Electrophoresis, whole protein in bacillus amyloliquefaciens stable phase somatic cells is divided Cloth is analyzed, and filters out the regulatory factor relevant to fragrant shepherd's purse element synthesis, contributes to from molecular level raw for the fragrant shepherd's purse element of transformation Produce bacterial strain and reliable information is provided.
But, the method for existing Two-Dimensional Gel Electrophoresis, in carrying out stable phase somatic cells, the distribution of whole protein is entered When row is analyzed, it is analyzed especially for the distribution of whole protein in bacillus amyloliquefaciens stable phase somatic cells, exists Two-dimensional Gel Electrophoresis resolution bad problem the highest, repeated.
Summary of the invention
It is an object of the invention to provide a kind of two-dimensional electrophoresis method for protein and application thereof, solve existing protein double To the method for electrophoresis, when in carrying out stable phase somatic cells, the distribution of whole protein is analyzed, especially for starch liquefacation In bacillus cereus stable phase somatic cells, the distribution of whole protein is analyzed, exist Two-dimensional Gel Electrophoresis resolution the highest, repeat The problem that property is bad.
The invention provides a kind of two-dimensional electrophoresis method for protein, including:
Step 1, prepares protein example；
Step 2, isoelectric focusing electrophoresis, specifically include:
Utilize hydrating fluid that adhesive tape is carried out aquation, obtain the adhesive tape that aquation is good, and good adhesive tape pH of aquation is 4-7, wherein Containing the carbamide of following components: 41-42.5g, the thiourea of 15-16g, CHAPS, 0.9-of 2-2.2g in every 100mL hydrating fluid The bromjophenol blue of IPG Buffer, 0.002-0.003g of DTT, 2-2.2g of 1.1g, and the solvent of hydrating fluid is ddH2O；
The adhesive tape that aquation is good is contacted with electrophoresis equipment, isoelectric focusing electrophoresis service condition is set, and carries out protein sample The loading of product, finally carries out isoelectric focusing electrophoresis, obtains the adhesive tape focused on, and wherein voltage and the time of isoelectric focusing electrophoresis adopts Arrange with following: S1:50v, 10h；S2:250v, 3h；S3:500v, 3h；S4:1000v, 1h；S5:8000v, 3h；S6:8000v, 6-6.5h；
Step 3, is balanced the adhesive tape obtaining having focused on, and then carries out sds page vertical electrophoresis, To the running gel containing protein；
Step 4, uses the method for silver staining dyeing to dye the running gel containing protein, the two-way electricity of final acquisition Swimming gel.
Preferably, in step 1, prepare protein example and specifically implement according to following steps:
Step a, collects the somatic cells of stable phase；
Step b, takes the lysate of certain volume, adds somatic cells by the concentration of 0.1-0.2g/mL, and presses 1mg/mL's Concentration adds compound protein enzyme inhibitor, and the mixed liquor obtained is placed in ice bath, sonicated cells while ice bath, until mixed Close liquid and become clarification, ultrasonic after, add nuclease mixture by the concentration of 10 μ L/mL, room temperature is placed, finally centrifugal and collect Supernatant, obtains whole protein sample.
Preferably, described step b is specifically implemented according to following steps:
Take 5mL lysate, add 0.5-1.0g thalline carefully, and add 1mg compound protein enzyme inhibitor, the mixing that will obtain Liquid is placed in ice bath, while ice bath under 80hz frequency sonicated cells, until mixed liquor become clarification；
After ultrasonic, adding 50 μ L nuclease mixture, room temperature places 1h；
13000 × g, 4 DEG C of centrifugal 30min, collect supernatant, obtain holoprotein sample.
Preferably, described adhesive tape is the IPG adhesive tape of 24cm, and the applied sample amount of protein example is 200 μ g.
Preferably, when the voltage of described isoelectric focusing electrophoresis is arranged, S6:8000v, 6h.
Present invention also offers a kind of two-dimensional electrophoresis method for protein, at detection bacillus amyloliquefaciens stable phase thalline The application of whole protein distribution in cell.
A kind of two-dimensional electrophoresis method for protein that the present invention provides, carrying out, bacillus amyloliquefaciens stable phase thalline is thin When in born of the same parents, the distribution of whole protein is analyzed, Two-dimensional Gel Electrophoresis resolution is high, reproducible.By the method for the present invention, right In bacillus amyloliquefaciens stable phase somatic cells, the distribution of whole protein is analyzed, and filters out relevant to fragrant shepherd's purse element synthesis Regulatory factor, contributing to producing bacterial strain for the fragrant shepherd's purse element of transformation from molecular level provides reliable information, has bigger economy It is worth and social meaning.
Accompanying drawing explanation
Fig. 1 is bacillus amyloliquefaciens CGMCC5569 and the Two-dimensional Gel Electrophoresis of bacillus amyloliquefaciens FMB50；
Fig. 2 is the percent profile schematic diagram of the cell location of differentially expressed protein；
Fig. 3 is the result figure of the relative expression quantity utilizing qRT-PCR analysis comA and spo0A；
Fig. 4 is comA and the imaginary schematic diagram of the fragrant shepherd's purse element route of synthesis of spo0A regulation.
Detailed description of the invention
The present invention is described in detail with detailed description of the invention below in conjunction with the accompanying drawings, it is to be understood that the protection of the present invention Scope is not limited by detailed description of the invention.
The invention provides a kind of two-dimensional electrophoresis method for protein, thin at detection bacillus amyloliquefaciens stable phase thalline During the application that in born of the same parents, whole protein is distributed, specifically implement according to following steps:
Step 1, prepares protein example
Step 1.1, collects the somatic cells of bacillus amyloliquefaciens stable phase
Bacillus amyloliquefaciens is cultivated to stable phase, in 6000 × g, and 4 DEG C of centrifugal 5min, collection precipitate, and rapidly Tris-HCl washing precipitate 3 times with the 20mmol/L being cooled to 4 DEG C in advance, it is thus achieved that somatic cells, wherein the pH of Tris-HCl is 6.8。
It should be noted that bacillus amyloliquefaciens cultivates about 36h i.e. up to stable phase；Wash with Tris-HCl every time After washing precipitate, all in 6000 × g, 4 DEG C of centrifugal 5min；The somatic cells obtained is in liquid nitrogen flash freezer, and-70 DEG C save backup.
Step 1.2, extracts the whole protein in the somatic cells of bacillus amyloliquefaciens stable phase, obtains holoprotein sample Product
Take 5mL lysate, add 0.5-1.0g thalline carefully, and add 1mg compound protein enzyme inhibitor, the mixing that will obtain Liquid is placed in ice bath, while ice bath under 80hz frequency sonicated cells, until mixed liquor become clarification；After ultrasonic, add Entering 50 μ L nuclease mixture, room temperature places 1h；13000 × g, 4 DEG C of centrifugal 30min, collect supernatant, obtain holoprotein sample Product, can put into-70 DEG C by holoprotein sample and save backup, it is also possible to be directly used in two-dimensional electrophoresis.
1mg protease inhibitor and 10 μ L nuclease mixture, Ke Yichong is added in holoprotein Sample Preparation Procedure Code insurance demonstrate,proves quality and the quantity of extracted holoprotein.
It should be noted that the compound protein enzyme preparation that described compound protein enzyme inhibitor is Roche company, described nucleic acid Enzymatic mixture is the nuclease mixture that GE Healthcare buys.
It should be noted that every 100mL lysate comprises the carbamide of following components: 42.04g, the thiourea of 15.2g, 4g CHAPS (3-[3-(gallbladder amido propyl) dimethylamino] propane sulfonic acid inner salt), the DTT (DTT) of 1g, the IPG of 2g Buffer, the solvent of lysate is ddH2O.Wherein during lysate preparation, first carbamide, thiourea, CHAPS, IPG Buffer are mixed Even, add DTT before using.
It should be noted that in order to ensure different sample when waiting point focusing electrophoresis loading, the applied sample amount of protein is the same, Protein concentration is measured with 2-D quantification kit (GE Healthcare).Using 2mg/mL BSA as standard solution, use and divide Light photometric determination sample and titer absorptance under 480nm wavelength；According to the absorptance of standard solution protein content, Mark and draw standard curve；Sample is compared with standard curve, obtains sample protein concentration.
Step 2, isoelectric focusing electrophoresis
Utilize hydrating fluid that the IPG adhesive tape of 24cm is carried out aquation, obtain the adhesive tape that aquation is good, and good adhesive tape pH of aquation is 4-7, wherein containing the carbamide of following components: 41-42.5g, the thiourea of 15-16g, 2-2.2g in every 100mL hydrating fluid The bromjophenol blue of IPG Buffer, 0.002-0.003g of DTT, 2-2.2g of CHAPS, 0.9-1.1g, and the solvent of hydrating fluid is ddH2O；
It should be noted that wherein containing the carbamide of following components: 41-42.5g, 15-16g in every 100mL hydrating fluid Thiourea, the bromjophenol blue of IPG Buffer, 0.002-0.003g of DTT, 2-2.2g of CHAPS, 0.9-1.1g of 2-2.2g, and water The solvent changing liquid is ddH2O, all can realize technical scheme, reach the effect of the present invention, owing to remaining step is identical, The most only so that every 100mL hydrating fluid to contain the IPG of DTT, 2g of CHAPS, 1g of the carbamide of 42.04g, the thiourea of 15.2g, 2g Technical scheme is described as a example by the bromjophenol blue of Buffer, 0.002g.
The adhesive tape that aquation is good is contacted with electrophoresis equipment, isoelectric focusing electrophoresis service condition is set, and carries out protein sample The loading of product, finally carries out isoelectric focusing electrophoresis, obtains the adhesive tape focused on, and wherein voltage and the time of isoelectric focusing electrophoresis adopts Arrange with following: S1:50v, 10h；S2:250v, 3h；S3:500v, 3h；S4:1000v, 1h；S5:8000v, 3h；S6:8000v, 6h；
It should be noted that S6:8000v, 6-6.5h all can realize technical scheme, reach the effect of the present invention Really, owing to remaining step is identical, the most only with S6:8000v, technical scheme is described as a example by 6h.
Treat that 24cm adhesive tape isoelectrofocusing reaches 80000vhr, time, isoelectric focusing electrophoresis terminates.
During it should be noted that hydrating fluid is prepared according to the following steps, first by carbamide, thiourea, CHAPS, IPG Buffer, bromine Phenol orchid and ddH2O mixes, and-20 DEG C save backup, and adds DTT before using.
It should be noted that S1 is low-voltage hydration step, S2 is boosting step.
It should be noted that different length can be taken or takes the adhesive tape of varying number and carries out electrophoresis, the present embodiment simultaneously The method of the present invention is explained by the IPG adhesive tape that simply have chosen the most excellent length.
Step 3, is balanced the adhesive tape obtaining having focused on, and then carries out sds page vertical electrophoresis, To the running gel containing protein, specifically include:
Step 3.1, DTT balances: the adhesive tape focused on is taken out and puts in balance pipe, the seating surface patch of the adhesive tape focused on Inboard wall of test tube, adds the slowly shake balance 15min on shaking table of the level pad containing 2%DTT, and wherein 2%DTT refers to put down In weighing apparatus buffer, DTT mass concentration (w/v) is 2%, and the level pad of this step uses the conventional balance in this area and delays Rush liquid.
Step 3.2, IAA (iodoacetamide) balances: the adhesive tape balanced in step 3.1 is taken out and is put into SDS running buffer Be stained with gently in liquid several under, be put in the balance liquid containing 2.5%IAA lucifuge balance 15min, finally electrophoretic buffer in SDS slightly Micro-cleaning, filter paper blots excessive moisture, and during wherein 2.5%IAA refers to level pad, IAA mass concentration (w/v) is 2.5%.
It should be noted that in SDS electrophoretic buffer containing 3.03% Tris-base, the glycine of 14.4%, 1% SDS, and SDS electrophoretic buffer solvent is ddH2O。
Step 3.3, sds page vertical electrophoresis
Step 3.3.1, compound concentration is the homogeneous sds polyacrylamide separating gel of 12.5%.
In the sds polyacrylamide separating gel of every 100mL 30% acrylamide gel containing 41.7mL store liquid, The pH of 25mL is the ddH of 10% (w/v) SDS, 31.8mL of 1.5M Tris-HCl, 1mL of 8.82O and the 10% (w/ of 500 μ L v)APS；During the preparation of sds polyacrylamide separating gel, first acrylamide gel is stored liquid, Tris-HCl, SDS and ddH2O Mixing, magnetic agitation is allowed to fully mix, and vacuum suction 30min adds APS.The SDS polyacrylamide of every 100mL before using Amine separating gel adds the TEMED of 33 μ L, mixing, then start encapsulating, glue surface covers a little saturated n-butyl alcohol, before use SDS electrophoretic buffer rinses glue surface three times, and acrylamide gel at least solidifies and reaches 3h and just can use.
Step 3.3.2, is placed in sds polyacrylamide separating gel top by the IPG adhesive tape balanced, and gets rid of gas Bubble, makes IPG adhesive tape be in close contact with gel surface, does not make adhesive tape face and contact glass sheet, the most do not move back and forth in operation Adhesive tape, by low melting-point agarose sealing fluid-tight adhesive tape, drains the bubble between adhesive tape and gel face.
Step 3.3.3, upper Marker: first in 95 DEG C, middle low molecular weight protein (LMWP) Marker is boiled 5min, draws Marker and drips On the filter paper of length and width about 1mm, filter paper is placed in the alkaline end of adhesive tape, it should be noted that middle low molecular weight protein (LMWP) Marker Molecular weight ranges be 7.1-209KD.
Step 3.3.4, after 10 × SDS electrophoretic buffer is diluted to 1 × SDS electrophoretic buffer, adds in electrophoresis tank, to the greatest extent Amount is avoided producing too much bubble.After low melting-point agarose sealing liquid solidifies, gel is transferred to Vertial electrophorestic tank, needs explanation , 10 × SDS electrophoretic buffer configures according to a conventional method, wherein containing 30.3g in every 100mL10 × SDS electrophoretic buffer The glycine of Tris-base, 144.2g, the SDS of 10g, and 10 × SDS electrophoretic buffer solvent is ddH2O。
Step 3.3.5, switches on power, and carries out electrophoresis with Ettan-Dalt II vertical electrophoresis apparatus, and temperature controls at 20 DEG C, Invariable power, 24cm adhesive tape parameter is set to P1 2w/ glue, 1h；P2 5w/ glue 30min；P3 15w/ glue 10h.In bromjophenol blue leading edge When running to gel bottom margin, terminating electrophoresis, the 24cm adhesive tape vertical electrophoresis operation time is 7h.
Step 4, uses the method for silver staining dyeing to dye the running gel containing protein, the two-way electricity of final acquisition Swimming gel
Step 4.1, takes out the gel slab after step 3.3.5 electrophoresis, peels off gel.
Step 4.2, fixing: gel is placed in fixative fixes 3h or overnight, the compound method of every 500mL fixative is: 50mL glacial acetic acid, 200mL dehydrated alcohol, add deionized water and be settled to 500mL.
Step 4.3, sensitization: use sensitizing solution, sensitization 30min, the compound method of every 500mL sensitizing solution instead after fixing end For: 150mL dehydrated alcohol, 1g sodium thiosulfate, 34g anhydrous sodium acetate, add deionized water dissolving and be settled to 500mL.
Step 4.4, washing: pour out sensitizing solution, to wash 3 times with deionization, each washing time is 5min.
Step 4.5, silver staining: utilize silver staining liquid silver staining 20min, the compound method of every 500mL silver staining liquid to be: silver nitrate 1.25g, adds deionized water dissolving and is settled to 500mL.
Step 4.6, washing: after pouring out silver staining liquid, to wash 1 time with deionization, washing time is 2min.
Step 4.7, colour developing: examine gel colour developing situation after adding nitrite ion, until protein spots is revealed as completely Only, about the 5-10min, the compound method of every 500mL nitrite ion is the most about needed to be: 12.5g natrium carbonicum calcinatum adds deionized water Being settled to 500mL after dissolving, adding 200 μ L volume fractions in every 500mL nitrite ion before use is the formaldehyde of 37%.
Step 4.8, terminates: after pouring out nitrite ion, add stop buffer, color development stopping, the preparation of every 500mL stop buffer immediately Method is: 7.3g EDTA-Na2Add deionized water and be settled to 500mL.
Step 4.9, preserves: wash 2 times with deionization after terminating 10min, it is thus achieved that two-dimensional electrophoresis gel, each washing time For 10min.
In order to study bacillus amyloliquefaciens CGMCC5569 (culture presevation number of this bacterial strain is CGMCC5569) and sweet smell Whole protein distribution situation in shepherd's purse element superior strain bacillus amyloliquefaciens FMB50 (culture presevation number is CGMCC6249), looks for Go out the protein distributional difference between two bacterial strains, filter out the regulatory factor relevant to fragrant shepherd's purse element synthesis, utilize the present invention's Method carries out two-dimensional electrophoresis to the whole protein in bacillus amyloliquefaciens CGMCC5569 and FMB50 respectively, and to two-dimensional electrophoresis Gel carries out image acquisition and atlas analysis, specifically includes:
Step A, the two-dimensional electrophoresis gel ImageScanner TM scanner after dyeing carries out transmission scan, digitized Image file uses ImageMasterR 2D Elite (Version 2.00) software of Amersham Pharmacia company to divide Analysis.Image analysis process includes the coupling (Match) of the detection of protein spots, quantization, background deduction and point.Protein spots is automatic Detect laggard edlin, first therefrom choose one piece of gel images before protein spots coupling as with reference to glue (Reference gel), And set up some match points (Seed matches), then other protein spots of Auto-matching, with the point not matched that with reference to glue It is added into reference in glue.3 glue one repeating groups (Replicate groups) of composition of same sample, differential expression egg The expression intensity of white matter Student-t inspection statistics is analyzed.Selecting reproducible and have statistical significance, expression differs Point more than twice does Mass Spectrometric Identification.
Fig. 1 is bacillus amyloliquefaciens CGMCC5569 and the Two-dimensional Gel Electrophoresis of bacillus amyloliquefaciens FMB50, Wherein Figure 1A is the Two-dimensional Gel Electrophoresis of bacillus amyloliquefaciens CGMCC5569, and Figure 1B is the Two-dimensional Gel Electrophoresis of FMB50.
As shown in Figure 1, with ImageMasterR 2D Elite (Version 2.00) software to two obtained strain bacterial strains The Two-dimensional Gel Electrophoresis of full cell soluble protein is analyzed, and the Abundances being found to have 50 protein sites there occurs change.Root According to the match condition of Two-dimensional Gel Electrophoresis under different condition, to reproducible in gel pattern, more than 2 times are occurred to become Abundances 50 protein spots changed have carried out Mass Spectrometric Identification, it is thus achieved that 50 complete peptide fingerprinting spectrums, further Mascot data Library searching shows, wherein protein site 87 and 433,115 and 132,443 and 446,461 and 493, and 473 and 501,508 and 697 is same One albumen, altogether 44 protein of successful identification.37 protein expressions are had to raise in bacillus amyloliquefaciens FMB50,4 eggs White down-regulated expression, 5 albumen only occur in bacillus amyloliquefaciens CGMCC5569, and 4 albumen are only at starch liquefacation spore Bacillus FMB50 occurs.
Step B, film dosim, Mass Spectrometric Identification
Step B1, protein spots enzyme action
Step B1.1, apparatus needed for alcohol disinfecting.
Step B1.2, deionization washing glue 3 times, each washing time is 10min.
Step B1.3, takes from glue a little with diameter 1.5mm rifle head, is taken by point of destination whole as far as possible.
Step B1.4, the protein spots of taking-up is placed in 96 orifice plates, and every hole adds deionized water 50 μ L, and ultrasound wave is (ultrasonic Power 20W, working time 60s) process 3 times, each process time is 10min.
Step B1.5,3000rpm is centrifuged 1min, after suck dry moisture, adds acetonitrile (ACN) 50 μ L, vortex 5min.
Step B1.6,3000rpm is centrifuged 1min, blots ACN, is subsequently adding 10mM DTT/25mM Ammonium bicarbonate food grade 50 μ L, and 56 DEG C water-bath 1h.
Step B1.7,3000rpm is centrifuged 1min, blots, and adds 55mM IAA/25mM Ammonium bicarbonate food grade 50 μ L, is placed in darkroom 45min。
Step B1.8,3000rpm is centrifuged 1min, blots, and adding volume fraction is the ACN of 50%, is dehydrated step by step, after blotting Naturally dry 5min, add pancreatin/25mM Ammonium bicarbonate food grade 2 μ L after 5ng/ μ L sequence modification, place 30min for 4 DEG C.
Step B1.9, adds 25mM Ammonium bicarbonate food grade 7.5 μ L, places 12h for 37 DEG C.
Step B1.10, adds the TFA that 0.35 μ L volume fraction is 2% and terminates.
Step B2, mass spectrum sample point sample
Step B2.1, before point sample, apparatus used all disinfects in alcohol.
Step B2.2, washes target: the 50% ultrasonic 10min of methanol that volume fraction is, 100% methanol ultrasonic (5MHz, 20w) 10min, dries.
Step B2.3, sample treatment before point sample: add the TFA that volume fraction is 0.5% in the sample cell drained and dissolve Mixtures of polypeptides.
Step B2.4, the preparation of substrate: alpha-cyano-beta-hydroxy cinnamic acid (HCCA) is dissolved in containing 30%ACN/ 0.1%TFA is made into saturated solution, fully high speed centrifugation after vibration.
Step B2.5, point sample: by sample and substrate by drawing 1 μ L point sample after 1: 1 equal-volume mixing on point sample target, be dried Dry.
Step B2.6, the detection of peptide mass fingerprinting spectrum: carry out spectrogram collection, laser under postponing desorbing and reflective-mode Wavelength 337nm, accelerating potential arranges 19KV.Each peptide spectrogram is about being added by 200 bombardment bases forming, and external standard uses cloth Shandong Gram company's peptide mixer standard substance, internal standard uses tryptic peak of autotomying, respectively m/z 842.50 and m/z 2211.10.
Step B2.7, washes target: after detection terminates, inhale and abandon after washing target 30-60s with the TFA that volume fraction is 0.1%.
Step C, the peptide fragment mass fingerprint obtaining step B carries out data base querying
Peptide mass fingerprinting spectrum refers to that protein is mixed by the fragments of peptides produced after the highest proteolytic enzymes hydrolize of specificity Compound quality collection of illustrative plates.Owing to each protein amino acid sequence is different, so with the peptide sheet obtained after proteolytic enzymes hydrolize Section mixture also differs, and the mass number of its peptide fragment mixture also has characteristic, referred to as peptide mapping fingerprinting (PMF), available Qualification in protein.Compared by peptide mapping fingerprinting theoretical in the peptide mapping fingerprinting that experiment is obtained and data base Protein just can be identified after relatively.
With Mascot search engine (http://www.matrixscience.com//mascot/cgi/search_ form.pl？FORMVER=2&SEARCH=PMF) retrieval of data base is carried out.Search condition: Protein Data Bank selects NCBInr storehouse, source of species selects other firmicutes, and enzyme selects Trypsin, it is allowed to incomplete enzymatic fragment selects Being 1, the fragments of peptides molecular weight limits of error are 120ppm, and ion is chosen as MH+, fix and are modified to Carbamidomethyl (C), variable is modified to Oxidation (M).
Step D, the protein identifying step C carries out bioinformatic analysis
Compare identified protein spots isoelectric point, IP in gel, isoelectric point, IP that molecular weight values obtains with experiment, molecular weight, Analyze the situation that meets therebetween and possible post translational modification situation.Utilize protein ortholog bunch (COG, http: // Www.ncbi.nlm.nih.gov/COG/), experimental identification protein is carried out function classification.According to UniProKB (www.uniprot.org) bacillus amyloliquefaciens protein information during data base and relevant document are reported, obtains albumen The biological process of matter participation and molecular function.According to PSORTb Version 3.0.1 software (http: // Www.psort.org/psortb/index.html) experimental identification protein is carried out cell positioning analysis.
Embodiments of the invention amount to identifies successfully 44 protein, with PSORTb Version 3.0.1 software (http://www.psort.org/psortb/index.html) carries out cell positioning analysis to it.Wherein there are 39 albumen fixed Position is at Cytoplasm, and 1 protein localization is outside born of the same parents, and 1 protein localization is at cell membrane, and 4 albumen the unknown cells position.Fig. 2 is for poor The percent profile schematic diagram of the cell location of different expressing protein, illustrates the albumen that the protein separated is mainly in Cytoplasm Matter, accounts for the 86.36% of the total protein being separated to.
(comA and spo0A is for relevant to fragrant shepherd's purse element synthesis to utilize qRT-PCR to analyze the relative expression quantity of comA and spo0A Gene), result is as it is shown on figure 3, comA and spo0A expression of mRNA in FMB50 is respectively it at starch liquefacation spore In bacillus CGMCC5569 15.8 times and 12.1 times of expression.It is consistent with protein level in the expression of transcriptional level.According to reality Testing results presumption, in bacillus amyloliquefaciens, comA and spo0A regulatory factor is by activating fragrant shepherd's purse element synthase gene fen Thus fragrant shepherd's purse element has been synthesized positive regulating and controlling effect (seeing Fig. 4).
The two-dimensional electrophoresis system that the present invention sets up, improves 2-DE method, respectively to bacillus amyloliquefaciens egg The committed steps such as the selection of the preparation of white matter sample, applied sample amount, colouring method, adhesive tape pH and 2-DE parameter are optimized.Its Optimize postcondition as follows: colouring method selects silver staining；Adhesive tape pH value is 4-7；24cm adhesive tape applied sample amount is 200 μ g.
, using containing 7M carbamide and the hydrating fluid of 2M thiourea, the isoelectrofocusing in conjunction with low-voltage desalination can obtain height meanwhile Resolution, reproducible protein 2-DE collection of illustrative plates.Through software analysis after silver staining, individual 2-DE collection of illustrative plates can detect 1000 Above protein site.
The present invention utilizes methods analyst bacillus amyloliquefaciens CGMCC5569 and the starch liquefacation of comparative proteome The protein expression difference of bacillus cereus FMB50.Based on above-mentioned experiment, have studied the solubility whole protein of gene FMB50 The change of 2-DE collection of illustrative plates, and the protein site of differential expression is carried out mass spectral analysis.2-DE pattern analysis results shows: at FMB50 In, through Software match after gel silver staining, the wherein Plantago fengdouensis 50 differential protein spots more than 2 times are carried out MALDI-TOF- MS analyzes, and has 44 protein sites effectively to be identified.Wherein there are 2 kinds of regulatory factors of ComA and Spo0A and fragrant shepherd's purse element synthesis phase Close.
The present invention protein to being identified is reported its function by COG ortholog bunch website and relevant document Be classified, find the function of these protein relate to metabolism, energy produce and convert, cell division and chromosome distribution, Translation, Ribosome Structure and biosynthesis, DNA replication dna, recombinate and repair, cell movement and secretion, post translational modification, general merit The aspects such as energy prediction.Wherein the protein of expliciting the position has 86.36% to be positioned in Cytoplasm.
Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation Property concept, then can make other change and amendment to these embodiments.So, claims are intended to be construed to include excellent Select embodiment and fall into all changes and the amendment of the scope of the invention.
Obviously, those skilled in the art can carry out various change and the modification essence without deviating from the present invention to the present invention God and scope.So, if these amendments of the present invention and modification belong to the scope of the claims in the present invention and equivalent technologies thereof Within, then the present invention is also intended to comprise these change and modification.

Claims (6)

1. a two-dimensional electrophoresis method for protein, it is characterised in that including:
Step 1, prepares protein example；
Step 2, isoelectric focusing electrophoresis, specifically include:
Utilize hydrating fluid that adhesive tape is carried out aquation, obtain the adhesive tape that aquation is good, and good adhesive tape pH of aquation is 4-7, the most often Containing the carbamide of following components: 41-42.5g, the thiourea of 15-16g, CHAPS, 0.9-1.1g of 2-2.2g in 100mL hydrating fluid The bromjophenol blue of IPG Buffer, 0.002-0.003g of DTT, 2-2.2g, and the solvent of hydrating fluid is ddH2O；
The adhesive tape that aquation is good is contacted with electrophoresis equipment, isoelectric focusing electrophoresis service condition is set, and carries out protein example Loading, finally carries out isoelectric focusing electrophoresis, obtains the adhesive tape focused on, and wherein voltage and the time of isoelectric focusing electrophoresis uses such as Lower setting: S1:50v, 10h；S2:250v, 3h；S3:500v, 3h；S4:1000v, 1h；S5:8000v, 3h；S6:8000v, 6- 6.5h；
Step 3, is balanced the adhesive tape obtaining having focused on, and then carries out sds page vertical electrophoresis, is contained There is the running gel of protein；
Step 4, uses the method for silver staining dyeing to dye the running gel containing protein, and the final two-dimensional electrophoresis that obtains is coagulated Glue.
Two-dimensional electrophoresis method for protein the most according to claim 1, it is characterised in that in step 1, prepares protein example Specifically implement according to following steps:
Step a, collects the somatic cells of stable phase；
Step b, takes the lysate of certain volume, adds somatic cells by the concentration of 0.1-0.2g/mL, and presses the concentration of 1mg/mL Add compound protein enzyme inhibitor, the mixed liquor obtained is placed in ice bath, sonicated cells while ice bath, until mixed liquor Become clarification, ultrasonic after, add nuclease mixture by the concentration of 10 μ L/mL, room temperature is placed, finally centrifugal and collect Clearly, whole protein sample is obtained.
Two-dimensional electrophoresis method for protein the most according to claim 2, it is characterised in that described step b is specifically according to following Step is implemented:
Take 5mL lysate, add 0.5-1.0g thalline carefully, and add 1mg compound protein enzyme inhibitor, the mixed liquor obtained is put While ice bath, ice bath under 80hz frequency sonicated cells, until mixed liquor become clarification；
After ultrasonic, adding 50 μ L nuclease mixture, room temperature places 1h；
13000 × g, 4 DEG C of centrifugal 30min, collect supernatant, obtain holoprotein sample.
Two-dimensional electrophoresis method for protein the most according to claim 1, it is characterised in that described adhesive tape is the IPG glue of 24cm Bar, and the applied sample amount of protein example is 200 μ g.
Two-dimensional electrophoresis method for protein the most according to claim 1, it is characterised in that the voltage of described isoelectric focusing electrophoresis When arranging, S6:8000v, 6h.
6. according to the two-dimensional electrophoresis method for protein described in any one of claim 1-5, steady at detection bacillus amyloliquefaciens The periodically application of whole protein distribution in somatic cells.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106596222A (en) * 2016-12-07 2017-04-26 长春长生生物科技有限责任公司 Method for preparing electrophoresis sample of escherichia coli
CN110133287A (en) * 2019-05-22 2019-08-16 上海碧云天生物技术有限公司 Protein Extraction solution and its application
CN115128148A (en) * 2022-05-18 2022-09-30 上海交通大学 Single-cell protein detection double-layer hydrogel and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101260146A (en) * 2008-04-24 2008-09-10 中国农业大学 Bidirectional electrophoresis method for malus plant protein
CN103278370A (en) * 2013-04-23 2013-09-04 山西农业大学 Method sutiable for two-dimensional electrophoresis extraction and separation of total protein of rat and mouse testicular tissue sample
CN103319566A (en) * 2013-06-26 2013-09-25 昆明理工大学 Method for extracting fungus total protein
CN105018514A (en) * 2015-06-18 2015-11-04 浙江大学 Construction method for efficient biosynthesis of streptomycete drugs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101260146A (en) * 2008-04-24 2008-09-10 中国农业大学 Bidirectional electrophoresis method for malus plant protein
CN103278370A (en) * 2013-04-23 2013-09-04 山西农业大学 Method sutiable for two-dimensional electrophoresis extraction and separation of total protein of rat and mouse testicular tissue sample
CN103319566A (en) * 2013-06-26 2013-09-25 昆明理工大学 Method for extracting fungus total protein
CN105018514A (en) * 2015-06-18 2015-11-04 浙江大学 Construction method for efficient biosynthesis of streptomycete drugs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JUNFENG ZHAO等: "Differential Proteomics Analysis of Bacillus amyloliquefaciens and Its Genome-Shuffled Mutant for Improving Surfactin Production", 《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106596222A (en) * 2016-12-07 2017-04-26 长春长生生物科技有限责任公司 Method for preparing electrophoresis sample of escherichia coli
CN110133287A (en) * 2019-05-22 2019-08-16 上海碧云天生物技术有限公司 Protein Extraction solution and its application
CN110133287B (en) * 2019-05-22 2021-07-30 上海碧云天生物技术有限公司 Protein extraction solution and application thereof
CN115128148A (en) * 2022-05-18 2022-09-30 上海交通大学 Single-cell protein detection double-layer hydrogel and preparation method and application thereof

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