CN104945466A - Buffer solution used for free flow electrophoresis - Google Patents
Buffer solution used for free flow electrophoresis Download PDFInfo
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- CN104945466A CN104945466A CN201410122467.3A CN201410122467A CN104945466A CN 104945466 A CN104945466 A CN 104945466A CN 201410122467 A CN201410122467 A CN 201410122467A CN 104945466 A CN104945466 A CN 104945466A
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- damping fluid
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Abstract
The invention discloses buffer solution used for free flow electrophoresis. The buffer solution used for the free flow electrophoresis contains hydroxypropyl methyl cellulose. The buffer solution used for the free flow electrophoresis can be used for obviously improving separation efficiency of existing free flow electrophoresis.
Description
Technical field
The present invention relates to a kind of free stream cataphoresis field, relate more specifically to the damping fluid used in free stream cataphoresis.
Background technology
The isolation technique of protein is the direction being badly in need of in protein research developing always.First this technical difficulty is the special physico-chemical character of protein molecule self, the abundance difference between the protein molecule of expressing in a specific biological sample very big (10
15-10
18); The size of protein molecule and the difference of surface charge are also great; In addition, the viscosity of most of protein molecule is very large, easily causes non-specific binding and precipitation.Two-dimensional electrophoresis is usually used in the separation of whole protein, but is suitable only for the separation of soluble protein, and cannot protein molecule that effectively isolated molecule amount is less or larger.High performance liquid chromatography is the another Main Means of protein separation, it possesses multidimensional operation, the feature that level of automation is high, simultaneously, directly can inject mass spectrograph by the polypeptide of reversed phase chromatography separation and do further qualification, be conducive to the analysis of large-scale protein expression spectrum.But the technology of high performance liquid chromatography lacks the effective separation to whole protein.And high-efficient liquid phase chromatogram technology is based upon in the clastotype of liquid-solid exchange, but protein molecule is difficult to avoid in the non-specific binding of solid phase surface, and this combination seriously have impact on the rate of recovery after protein separation.In brief, no matter based on gel electrophoresis, or the protein stripping technique based on liquid chromatography meets all far away effective separation of complex proteins mixture.
The proposition of free stream cataphoresis (Free Flow Electrophoresis FFE) concept, is filled with new vitality to a point analysis of variance for biomacromolecule.FFE carries out in a thin layer liquid matrix, is equivalent to two of two-dimensional electrophoresis process conformities to get up disposable completing, but without the need to the support of any fixing matrix.Exactly because sepn process completes substantially in the liquid phase, FFE can ensure that protein losses is reduced to minimum level, and with regard to Theoretical Calculation, the protein recovery of FFE can close to 100%.FFE possesses the separation capacity of larger protein concn scope, and according to Theoretical Calculation, protein concn can be effectively separated at 0.05-5mg/ml.Like this, FFE had both been suitable for comparatively meticulous protein analysis work, and can be competent in the preparative separation experiment of protein.No matter from the fervent demand of proteome research for isolation technique revolution, or with regard to the developing direction of the separation science representated by FFE technology, FFE is one to be worth making great efforts and the scientific domain of investment.But free stream cataphoresis sepn process is a very complicated dynamic process, wherein involved correlative factor much and mutually involves, as thermal convection, hydromeehanics distortion, electrodynamics distortion etc. all have an impact to the stability of object separation and repeatability.After the U.S., France, Japan, Russia and China successively for successfully completing the separation test of space free flow electrophoresis, and its efficient separating resulting shows, free stream cataphoresis will be played a greater and greater role at life science with improving of himself.Different from the space free flow electrophoresis under microgravity environment, do not have the support of solid phase to there will be sedimentation and convection phenomena under action of gravity in electrophoresis process, greatly reduce purity and the output of separation.Obviously, the development of FFE is just awaiting new design.
In recent years, the technologic innovation of free stream cataphoresis Design and manufacture emerges in an endless stream.Such as by disengagement chamber material and special process process to improve the efficiency of Thin-layer separation chamber heat exchange; The flowing of vertical oppositely damping fluid reduces thermal convection to the impact of segregational stability; Adopt laminar flow rate setting device to carry out steady flow condition, the both direction in laminar flow cross section reduces hydromeehanics distortion etc.But the change in free stream cataphoresis damping fluid is less, adopt be mixed with glycerine and amphotericeledrolyte TAE electrophoretic buffer.But in fact, electrophoretic buffer affects no less important to the separating effect of free stream cataphoresis.
In electrophoresis, adopt the electrophoretic buffer more close with sample rate will mention separation efficiency undoubtedly.If because sample and electrophoretic buffer have larger density variation, can cause sample, in electrophoresis process, sedimentation occurs; Different density also can cause sample stream different with separating medium rate travel.Therefore in actually operating, often adopt the mode of adding glycerine in electrophoretic buffer, reach the consistent in density of sample and electrophoretic buffer.But glycerine is easily attached in disengagement chamber wall and pipeline, to the cleaning of equipment with preserve unfavorable, and glycerine does not have the ability of electric charge mediation, often needs to add the electrophoresis liquid compositions such as TAE, TBE.And TAE, TBE are often with the specific conductivity higher than sample, the resolving power of total system is caused to decline.
In addition, the electrophoresis chamber size that electrophoresis adopts also can affect electrophoresis result to a certain extent.Such as electrophoresis chamber size is larger, and the separating effect caused due to convection current, thermodiffusion reason can be poorer.
Based on above analysis, in this area, need the novel free stream cataphoresis damping fluid that significantly can promote the separation efficiency of free stream cataphoresis in large-size electrophoresis chamber.
Summary of the invention
The object of the invention is to the novel free stream cataphoresis damping fluid proposing a kind of high efficiency separation albumen.
For reaching this object, the present invention by the following technical solutions:
In in first, the invention provides a kind of damping fluid for free stream cataphoresis, described damping fluid comprises Vltra tears.
The damping fluid of free stream cataphoresis of the present invention can also comprise amphotericeledrolyte.
In the damping fluid of free stream cataphoresis of the present invention, the concentration of described Vltra tears can be 0.35%-0.5%, is preferably 0.4%-0.45%.
In the damping fluid of free stream cataphoresis of the present invention, described damping fluid can comprise the amphotericeledrolyte of 1%, can form pH3-10, preferred 5-8, more preferably the cushion gradient of 6-7 under electrical forces effect.
In a second aspect, the invention provides a kind of method of free stream cataphoresis, described method comprises the damping fluid adopted according to first aspect.
In in the 3rd, the invention provides the application of damping fluid in albumen sepn according to first aspect.
In in the 4th, the invention provides the application of damping fluid in proteome research according to first aspect.
Beneficial effect:
(1) use in the electrophoresis process of free stream cataphoresis damping fluid of the present invention, indicator has separation configuration (see Fig. 1) in good order in disengagement chamber, this explanation is carried out in the process of electrophoresis in the free stream cataphoresis system of large-size scale, and every factors such as thermal convection, hydromeehanics distortion, electrodynamics distortion reach a relative balance;
(2) between the pH3-10 that biomolecules extensively exists, the electrophoretic buffer of the application of the invention can realize more careful separation (see Fig. 2) in the free stream cataphoresis system of large-size scale, this also means between pH3-10, and this electrophoretic buffer has better resolving power.
(3) damping fluid of the present invention is applicable to the free stream cataphoresis system of large-size scale, thus obtains the better free stream cataphoresis system that flux is higher simultaneously of resolving effect, effectively can improve the recall rate to trace protein sample.
Accompanying drawing is sketched
Fig. 1. use the separation configuration of indicator in the electrophoresis process of electrophoretic buffer of the present invention.
Fig. 2. use the pH separation efficiency comparative result of the electrophoresis process of electrophoretic buffer of the present invention.
Fig. 3. use the results contrast of electrophoretic buffer of the present invention and business damping fluid Separation of Bovine serum albumin, chicken ovalbumin and human transferrin respectively.
Detailed Description Of The Invention
Buffer solution of the present invention is Vltra tears (HPMC).Vltra tears is a kind of non-ionic type mixed cellulose ethers, is called for short HPMC.Molecular-weight average 243.96.White or off-white powder.HMPC has hot gel property, i.e. cold water solubles, and (higher than gelling temp) is insoluble and form gel in the hot water, stablizes alkali and acid, unaffected in pH value 2 ~ 12 scope.Certain density HPMC has certain viscosity, has the density higher than water, effectively can prevent the sedimentation phenomenon of protein soln.And it has hot gel property, can increase in electrophoresis exothermal process medium viscosity, the electrophoresis zone distortion effectively preventing hydrokinetics to bring.More of paramount importance, namely HPMC can keep stable under different pH, and this makes after adding amphotericeledrolyte, can form stable high-resolution pH gradient, make the point focusing such as protein passes through be able to effective separation in disengagement chamber.
HPMC concentration optimization
In colloidal solution, when the density of disperse phase is greater than the density of dispersion medium, can settlement action be there is in diffusion process, and contrary to disperse phase density is less than dispersion medium density, often there is Float upward function.So we should select HPMC solution higher than protein soln density as electrophoretic buffer in theory.But when HPMC concentration increases, the resistance of electrophoretic buffer can increase on the one hand, produces larger joule heating, even may cause the hot gelatin phenomenon of HPMC.On the other hand, spring up at disengagement chamber the difference that process Midst density difference can cause the speed of springing up, finally cause the distortion in sample separation face.So we will need by optimized choice when HPMC concentration, and HPMC is 0.1-0.2% as suspending agent working concentration generally.In the present invention, in the concentration range of 0.3%-1.5%HPMC, have selected optimal concentration.
Amphotericeledrolyte concentration optimization
Amphotericeledrolyte effect, be that to form stable pH interval being separated in electric field, can be separated according to iso-electric point difference to make protein.No matter and stablize the formation in pH interval, or the separation of protein, all need enough focal times.Conductance reduces gradually in this process, and resistance raises gradually, after reaching certain voltage, realizes the separation of sample.Discussed before us, conductance difference between sample and damping fluid is less, the most applicable from electrofluid angle, but often containing certain salt in protein soln, if pursue close conductance, may need to use higher amphotericeledrolyte concentration, the point focusing such as so just needs the time more grown could form stable pH region.Because of between these point focusing separating effect and changes of electrofiuid mechanics, best point of the two balance another must be found, therefore after determining HPMC concentration, to amphotericeledrolyte working concentration be optimized.
Technical scheme of the present invention is further illustrated by embodiment below in conjunction with accompanying drawing.
Embodiment 1
Weigh 3.5g HPMC, add 1000ml deionization pure water, after fully dissolving, be 0.35%HPMC solution.Get the amphotericeledrolyte (SERVA) of 5ml pH3-10, add 495ml0.35%HPMC solution, the electrophoresis liquid after fully dissolving is the isoelectric focusing electrophoresis damping fluid after the present invention's optimization.This institute electrophoresis chamber size: 64cmX18cmX0.5mm.
Embodiment 2
Weigh 5g HPMC, add 1000ml deionization pure water, after fully dissolving, be 0.5%HPMC solution.Get the amphotericeledrolyte (SERVA) of 5ml pH3-10, add 495ml0.5%HPMC solution, the electrophoresis liquid after fully dissolving is the isoelectric focusing electrophoresis damping fluid after the present invention's optimization.
Embodiment 3
Weigh 4g HPMC, add 1000ml deionization pure water, after fully dissolving, be 0.4%HPMC solution.Get the amphotericeledrolyte (SERVA) of 5ml pH3-10, add 495ml10.4%HPMC solution, the electrophoresis liquid after fully dissolving is the isoelectric focusing electrophoresis damping fluid after the present invention's optimization.
Embodiment 4
Damping fluid obtained in embodiment 1,2,3 is applied to the separation of protein solution, obtains similar result.As shown in Figure 3: by using this damping fluid, chicken ovalbumin, by originally there are 4 components, is concentrated into 2 components, and bovine serum albumin was concentrated into 4 main ingredients by originally there are 8 components.Human transferrin also has similar phenomenon.What is more important, by using this electrophoretic buffer, making original bovine serum albumin and the most component of chicken ovalbumin be all overlapping, becoming and be present in diverse component, thus achieve effective separation.
Preparation of samples: get 5mg chicken egg white, bovine serum albumin, human transferrin respectively, adds 1ml deionization pure water, and fully concussion mixing is until solution is thoroughly clarified.
Operation steps: instrumentation concrete steps are with reference to free stream cataphoresis instrument process specifications.Wherein loading 1mg albumen, loading flow velocity is 25rpm, and running buffer flow velocity is 50rpm.Arranging voltage is 1.25 kilovolts, and maximum current is restricted to 50 milliamperes, each component solution after 10 as a child collection isoelectrofocusing.
Claims (8)
1. for freely flowing a damping fluid for isoelectric focusing electrophoresis, it is characterized in that, described damping fluid comprises Vltra tears.
2. damping fluid according to claim 1, is characterized in that, described damping fluid also comprises amphotericeledrolyte.
3. damping fluid according to claim 1 and 2, is characterized in that, the concentration of wherein said Vltra tears is 0.35%-0.5%, is preferably 0.4%-0.45%.
4. damping fluid according to any one of claim 1 to 3, is characterized in that, wherein said amphotericeledrolyte concentration be 1%-2%, be preferably 1%.
5. damping fluid according to any one of claim 1 to 4, is characterized in that, the pH of described damping fluid is 3-10, preferred 5-8, more preferably 6-7.
6. a method for free stream cataphoresis, described method comprises employing damping fluid according to any one of claim 1 to 5.
7. the application of damping fluid according to any one of claim 1 to 5 in albumen sepn.
8. the application of damping fluid according to any one of claim 1 to 5 in proteome research.
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| CN201410122467.3A CN104945466A (en) | 2014-03-28 | 2014-03-28 | Buffer solution used for free flow electrophoresis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113563412A (en) * | 2021-06-23 | 2021-10-29 | 上海蓝析生物技术有限公司 | Free-current isoelectric focusing electrophoresis buffer system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008155420A1 (en) * | 2007-06-20 | 2008-12-24 | Becton, Dickinson & Company | Ffe media and ffe methods comprising volatile separation media |
| CN101512334A (en) * | 2006-06-20 | 2009-08-19 | 贝克顿迪肯森公司 | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
| WO2009133153A1 (en) * | 2008-04-29 | 2009-11-05 | Becton, Dickinson And Company | Methods and systems for the separation and analysis of analytes using ffe |
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2014
- 2014-03-28 CN CN201410122467.3A patent/CN104945466A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101512334A (en) * | 2006-06-20 | 2009-08-19 | 贝克顿迪肯森公司 | Method and device for separation and depletion of certain proteins and particles using electrophoresis |
| WO2008155420A1 (en) * | 2007-06-20 | 2008-12-24 | Becton, Dickinson & Company | Ffe media and ffe methods comprising volatile separation media |
| WO2009133153A1 (en) * | 2008-04-29 | 2009-11-05 | Becton, Dickinson And Company | Methods and systems for the separation and analysis of analytes using ffe |
Non-Patent Citations (2)
| Title |
|---|
| GERHARD WEBER: "Recent developments in preparative free flow isoelectric focusing", 《ELECFROPHORESIS》 * |
| PETER HOFFMANN: "Continuous free-flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: A non two-dimensional gel electrophoresis-based proteome analysis strategy", 《PROTEOMICS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113563412A (en) * | 2021-06-23 | 2021-10-29 | 上海蓝析生物技术有限公司 | Free-current isoelectric focusing electrophoresis buffer system |
| CN113563412B (en) * | 2021-06-23 | 2023-12-19 | 上海蓝析生物技术有限公司 | Free-flow isoelectric focusing electrophoresis buffer system |
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Application publication date: 20150930 |