CN107488208A - A kind of isoelectric focusing of carrier-free ampholytes and the method for separation - Google Patents
A kind of isoelectric focusing of carrier-free ampholytes and the method for separation Download PDFInfo
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Abstract
The invention discloses a kind of method of isoelectric focusing of carrier-free ampholytes and separation, belongs to protein sample separation analysis field.Methods described is using paper passage, acid solution, alkaline solution, both sexes sample, two electrodes and dc source composition loop, described two electrodes are inserted in acid solution and alkaline solution respectively, the paper passage is contacted with acid and alkaline solution respectively using its both ends after the wetting of any of acid solution or alkaline solution, described two electrodes are connected by wire with dc source, and both sexes sample is loaded directly into in acid solution or alkaline solution or on paper passage.Compared with prior art, the quick focusing and separation of both sexes sample can be achieved without free carrier ampholyte by the present invention, and separation component easily reclaims, and are easy to and other analysis methods are combined.It is more preferable with mass spectrum when using volatility weak acid and exemplary volatile weak base system.
Description
Technical field
The invention belongs to protein sample to separate analysis field, is related to a kind of isoelectric focusing of carrier-free ampholytes and divides
From method.
Background technology
Isoelectric focusing (isoelectric focusing, IEF) is to be directed to the electrophoresis point of the both sexes components with isoelectric point
From one of pattern with concentration, it is widely used in the sign of the separation of complex proteins, purification and isoelectric point.With pH gradient
Split tunnel on, under electric field action both sexes components according to the difference of isoelectric point to the migration of its pI identical pH position and it is real
Now focus on and separate.The method for forming pH gradient in the prior art is broadly divided into two kinds：One kind is by free carrier both sexes electricity
Matter (carrier ampholyte, CA) is solved, CA is the mixture of a kind of amphoteric compound, after applying voltage, has close grade for electricity
The mixture of the serial amphoteric compound at point interval migrates in the presence of electric field, forms corresponding pH gradient, amphiphatic molecule exists
It is able to focus on separation in this gradient.But not only price is high for carrier ampholyte, and with other analysis methods such as mass spectrum
Interference can be produced during combination.Another kind is to be based on IPG (immobilized pH gradient) adhesive tape, using acrylamide as base
Matter utilizes different pK ampholytic monomer, and the polymer with certain pH gradient is formed after crosslinking.But commercially available IPG adhesive tape is not
Only price is high, and time-consuming by completion IEF.In order to avoid the use of carrier ampholyte, researcher also attempted no-load
The isoelectric focusing method of body ampholytes, such as self-focusing, thermic pH gradient and electrolysis form pH gradient, but pH gradient is not
Quantitatively provide, system complex and cumbersome.
Since paper substrate analytical equipment is incorporated into microfluidic analysis field by Whitesides seminars in 2007 first,
Paper substrate micro-fluidic analytical equipment (Paper based analytical device, PAD) just arouses widespread concern.
PAD has the advantages that inexpensive, portable, consumption sample is few, prepares simple and good biocompatibility.The application of paper substrate micro-fluidic at present
Various fields, such as food security, environmental monitoring and clinical diagnosis are covered.
At present, it has been suggested that a kind of paper substrate isoelectric focusing and separation method based on carrier ampholyte.Due to the party
Method realizes the quick poly- of both sexes sample on paper substrate analytical equipment by means of carrier ampholyte using isoelectric focusing method
Burnt and separation.Therefore, not only cost is high for this method, and carrier ampholyte with other analysis methods (such as mass spectral analysis)
Interference can be produced during combination, is unfavorable for separating and recovering the mass spectral characteristi of component.
In summary, need badly and seek one kind without carrier ampholyte, the quick poly- of both sexes sample can be realized
Burnt and separation, separation component easily reclaims and the method for isoelectric focusing and separation beneficial to mass spectral characteristi.
The content of the invention
(1) technical problems to be solved
In order to solve the above mentioned problem of prior art, the present invention provide it is a kind of without free carrier ampholyte,
Can realize the quick focusing and separation of both sexes sample, separation component easily reclaim and beneficial to mass spectral characteristi isoelectric focusing and point
From method.
(2) technical scheme
In order to achieve the above object, the main technical schemes that the present invention uses include：
A kind of isoelectric focusing of carrier-free ampholytes and the method for separation, methods described are molten using paper passage, acidity
Liquid, alkaline solution, both sexes sample, two electrodes and dc source composition loop, described two electrodes insert acid solution respectively
In alkaline solution, the paper passage using any of acid solution and alkaline solution wetting after its both ends respectively with acidity
Solution and alkaline solution contact, described two electrodes are connected by wire with dc source, and both sexes sample is loaded directly into acidity
In solution or alkaline solution or on paper passage.
As carrier-free ampholytes isoelectric focusing and separation method preferred scheme, the acid solution it is dense
Spend for 0.1mM-1.0M, the concentration of the alkaline solution is 0.1mM-1.0M.
As the preferred scheme of the method for isoelectric focusing and the separation of carrier-free ampholytes, the acid solution is to wave
Hair property weak acid HAc solution or HCOOH solution, the alkaline solution are exemplary volatile weak base NH3·H2O solution.
As the preferred scheme of the method for isoelectric focusing and the separation of carrier-free ampholytes, in described two electrodes
Anode uses platinum electrode, and negative electrode uses platinum electrode.
As the preferred scheme of the method for isoelectric focusing and the separation of carrier-free ampholytes, the paper passage is use
The split tunnel that Fiber Materials are made.
As the preferred scheme of the method for isoelectric focusing and the separation of carrier-free ampholytes, the paper passage is glass
Tunica fibrosa, the length of the paper passage is 5-100mm, and width is 100 μm of -10mm.
As the preferred scheme of the method for isoelectric focusing and the separation of carrier-free ampholytes, the paper passage is put on
On electric-field intensity scope be 3-300V/cm.
(3) beneficial effect
Compared with prior art, advantage of the invention is specific as follows：
1st, the present invention adopts without a range of freedom or immobilization carrier ampholytes on paper substrate analytical equipment
The quick focusing and separation of complicated both sexes sample (protein sample) are realized with the isoelectric focusing method of carrier-free ampholytes,
Separation component easily reclaims, and not only reduces cost, and avoids carrier ampholyte and analyzing (such as matter with follow-up other
Spectrum analysis) it is combined the caused mass spectral characteristi for disturbing, being advantageous to separate and recover component.
2nd, both sexes sample is loaded directly into in acid anodic dissolution or alkaline cathode solution or on paper passage, having two aspects
Advantage：On the one hand both sexes sample can be made powered, after DC voltage is applied, is advantageous to work of the both sexes sample component in electric field
With lower migration；Another aspect simplified operation.
Brief description of the drawings
Fig. 1 is the structural representation of paper substrate analytical equipment in the embodiment of the present invention 1；
Fig. 2 is isoelectric focusing and the focusing of separation phycocyanin and bovine hemoglobin biased sample in the embodiment of the present invention 1
Electric current versus time curve figure in separating resulting and focusing separation process；
Signal after the completion of Fig. 3 is pH distribution maps in the embodiment of the present invention 1 on paper passage and focused on paper passage is strong
Spend distribution map；
Fig. 4 is the signal strength distribution map on the paper passage in the embodiment of the present invention 1；
Fig. 5 is the SDS-PAGE phenograms and mass spectrogram that component to be recycled is focused in the embodiment of the present invention 1；
Fig. 6 is the Modulatory character analysis chart of the present invention.
Embodiment
In order to preferably explain the present invention, in order to understand, below in conjunction with the accompanying drawings, by embodiment, to this hair
It is bright to be described in detail.
Embodiment 1
The present embodiment proposes not carry out algae indigo plant by carrier ampholyte on the opening paper substrate analytical equipment shown in Fig. 1
Albumen and the isoelectric focusing of bovine hemoglobin biased sample and the method for separation, this method are molten using paper passage, acid solution, alkalescence
Liquid (being illustrated by taking volatility weak acid, exemplary volatile weak base solution as an example), both sexes sample, two electrodes and dc source form back
Road.
Specifically, adding the HAc solution that concentration is 10mM in anode liquid storage tank, pH=4.3, contain matter in HAc solution
Measuring the HEC that volume fraction (i.e. HEC quality and the volume ratio of HAc solution, m/V) is 0.2%, (wherein, HEC is hydroxy ethyl fiber
Element, belong to nonionic surface active agent, for suppressing EOF.There is silicone hydroxyl on paper passage surface, negatively charged, causes paper passage
On solution positively charged, solution has the overall trend moved to negative pole, referred to as EOF.In electrophoretic analysis, when electric osmose flows through
When big, conventional surfactant is suppressed.), insertion platinum electrode is as anode.Added in cathodic reservoir comprising concentration and be
0.5mg/mL phycocyanins and the biased sample that concentration is 1.0mg/mL bovine hemoglobins, the matrix of the biased sample select concentration
For 10mM NH3·H2O solution (pH=9.8), insertion platinum electrode is as negative electrode.Paper passage uses above-mentioned HAc solution-wets,
The both ends of paper passage after wetting respectively with HAc solution and NH3·H2O solution contacts, and the electrode of negative and positive two passes through wire and direct current
Source is connected, and forms loop.Paper channel selecting glass fibre membrane, the size a length of 35mm, a width of 3mm of the paper passage.Apply electric field
Intensity is 100V/cm DC voltage, and in the presence of electric field, certain pH gradient is formd on paper passage.Above-mentioned paper passage
It is whole circuit is formed loop directly using the purpose of the HAc solution-wets of anode.Certainly, above-mentioned paper passage can also be adopted
Split tunnel is made with other Fiber Materials.
The principle of the present invention is as follows：Paper passage is soaked with acid supporting electrolyte, and acid sun is immersed at its both ends respectively
In pole supporting electrolyte solution and alkaline cathode electrolyte solution.After applying DC voltage, the OH of cathodic reservoir-To anode tap
Migration, the H of anode pool+To cathodic migration, neutralization reaction occurs when both meet, this neutralizes interface and taken place from cathode terminal,
And gradually moved to anode tap；Cathode terminal is to the region for neutralizing interface, due to OH-Migration can form the region that alkalescence is successively decreased, and deposit
Material be mainly OH-.Anode tap is to the region for neutralizing interface, due to H+Migration can form the acid region successively decreased, exist
Material be mainly H+.Neutralization reaction interface, existing material are mainly H2O, it is rendered as neutrality.H+Not only made by electric field
With migrating to paper passage, and EOF can be also pumped into paper passage, so neutralization reaction will not proceed to anode storage
Liquid pool.Because neutralization reaction generates the low water of electrical conductivity, current reduction, the electromigration of charge species weakens, and final system reaches
Stable pH gradient is formd to poised state and on paper passage.
Phycocyanin (isoelectric point is about 4.45) and bovine hemoglobin (isoelectric point is about 6.8) sample, which are in, is higher than its grade electricity
It is negatively charged in the sample substrate (pH 10) of point, migrated in the presence of electric field to anode, its isoelectric point is moved closer to when moving
PH positions electric charge is zero, and is focused on herein so that the sample of different isoelectric points is separated.
Mobile phone is fixed on the eyepiece stalk of stereomicroscope with geometrical clamp, taken pictures software Camera FV-5 using specialty
Observe whole focusing, setting is taken pictures at intervals of 10s, focuses on separating resulting as shown in Fig. 2 when being 590s between when taking,
Phycocyanin and bovine hemoglobin have obtained while have focused on and separate.It can be seen from current-time curvel, from photo opporunity 350s
Start, electric current declines and maintains very low level, about 30 μ A, and this system can long-time stable presence.
PH information on paper passage and phycocyanin and bovine hemoglobin are obtained below by following operating method
Separating degree.Specifically, both sexes components etc. are not carried out by carrier ampholyte on the opening paper substrate analytical equipment shown in Fig. 1
Electrofocusing and the method for separation, are tested, other experiment conditions are referring to aforesaid operations condition in the case of sample blank.With
5mm is spacing, pH information when characterizing photo opporunity 590s with accurate pH test paper on paper passage.Using five replicate experiments as
As a result, it is known that the pH scopes formed on paper passage are 4.3-9.8 (referring to Fig. 3).The obtained phycocyanins of Fig. 2 and ox blood is red
Focusedimage of the albumen in 590s is placed in this gradient, it is known that the pH positions that phycocyanin focuses on are about 4.5, with its isoelectric point
4.45 is close, and the pH positions that bovine hemoglobin focuses on are about 6.5, close with its isoelectric point 6.8.Focusedimage is passed through into Image
J processing, obtain the signal strength distribution map on paper passage (referring to Fig. 4).According to the signal strength distribution map on paper passage and divide
From degree R calculation formulaIn formula, tR2：The retention time of latter component, t in adjacent two peakR1：Adjacent two
The retention time of previous component, W in peak2、W1：The peak base at this adjacent two peak is wide, it is known that tR2=1435, tR1=958, W2=133,
W1=137, the separating degree R that phycocyanin and bovine hemoglobin is thus calculated is 3.53, shows that both have reached and divides completely
From (when separating degree is 1.5 in chromatogram it is believed that two components are kept completely separate).
After the completion of can focusing on, the phycocyanin and bovine hemoglobin focused on paper passage is reclaimed, removal process tool
Body is as follows：1st, the focal zone on paper passage during photo opporunity 590s shown in Fig. 2 is cut, be placed in two centrifuge tubes；2nd, it is every
In individual centrifuge tube plus deionized water vibration is dissolved；3rd, two centrifuge tubes are put into centrifuge to be centrifuged, supernatant is retained after centrifugation
Liquid, that is, it is recycled sample.By above-mentioned phycocyanin and the recovery sample of bovine hemoglobin respectively in the opening paper substrate shown in Fig. 1
Isoelectric focusing again on analytical equipment, moreover it is possible to which phycocyanin and bovine hemoglobin are isolated in focusing, it was demonstrated that using the present embodiment
Method can also realize the recovery of sample.
In order to verify that established method can separate mixed protein, paper passage is cut into five equal portions (such as Fig. 5 a by us
It is shown), and recovery component is placed in EP pipes, then vibrated and dissolved using loading buffer, heat denatured carries out SDS-
PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis, abbreviation SDS-PAGE)
Analysis.As shown in Figure 5 b, the 1st swimming lane is low molecular weight protein marker, and the 2nd to 6 swimming lane is followed successively by 1 to No. 5 recovery component,
7th swimming lane is phycocyanin and bovine hemoglobin mixture.According to the pH distributions on paper passage and the pI of model protein used, the
2nd, 5 and 6 swimming lanes should occur without protein band；Phycocyanin and bovine hemoglobin should respectively appear in the 3rd and the 4th swimming lane.It is existing
Document shows that phycocyanin contains two subunits of α and β, and both molecular weight respectively may be about 18kDa and 19kDa；Bovine hemoglobin
In vain containing the close subunit of four molecular weight, molecular weight is 15kDa or 16kDa.SDS-PAGE characterization results as shown in Figure 5 b,
3 swimming lanes occur molecular weight be about 18,19kDa band, the band that about 15kDa and 30kDa occurs in the 4th swimming lane (may be by
Incomplete in albuminous degeneration, existing document also has similar phenomenon, explain it is main be classified as that bovine hemoglobin purity is not high or denaturation not
Completely).SDS-PAGE electrophoresis results are consistent with the report in document, show that established method can realize egg mix simultaneously
White focusing and separation.
It was found from Fig. 5 b SDS-PAGE phenograms, there is protein band in only No. 2 and No. 3 recovery components, other times
Receive appearance of the component without protein band.So carrying out Matrix Assisted Laser Desorption Ionization-Time of Flight tandem mass spectrometer
(MALDI-TOF/TOF) when verifying that established system mass spectrum is good, we only choose No. 2 and No. 3 recovery components and carried out
Analysis.Acetic acid and ammoniacal liquor are volatile component, are mass spectrum friendly electrolyte.At can be without desalination when with mass spectrometry
Reason, direct injected detection.
We by No. 2 and No. 3 recovery components be placed in EP pipes, with distilled water vibrate dissolve, ultrasound take supernatant then with
Matrix SA (3,5- dimethoxy-4 's-hydroxycinnamic acid) is mixed, and MALDI-TOF/TOF is detected under linear model.For the ease of
Compare, we have also carried out MALDI-TOF/TOF analyses to the protein sample (solvent is water) without PAD-CAF-IEF.
There is document to illustrate that phycocyanin contains two subunits of α and β, molecular weight is respectively 18.188kDa and 19.220kDa.Such as Fig. 5 c institutes
Show, dark and light spectral line be respectively phycocyanin sample and No. 2 recovery components spectrogram, MALDI-TOF/TOF measurement results
It has been shown that, phycocyanin and No. 2 recovery components contain two subunits of 18.192kDa and 19.289kDa, and it is same thing to show both
Matter, and more meet with literature value.Similarly, there is document to show that bovine hemoglobin contains four subunits, molecular weight be about 15kDa or
16kDa.As fig 5d, dark and light spectral line be respectively bovine hemoglobin sample and No. 3 recovery components spectrogram, MALDI-
TOF/TOF measurement results show that bovine hemoglobin and No. 3 recovery components contain two Asias of 15.055kDa and 15.959kDa
Base, it is bovine hemoglobin to show No. 3, and measurement result and literature value are more consistent.
Embodiment 2-10
Similar with separation method with the isoelectric focusing of embodiment 1, embodiment 2-10 is molten by adjusting anodolyte HAc
The concentration (0.1mM-1M) of liquid, catholyte NH3·H2Concentration (0.1mM-1M), the length and width dimensions of paper passage of O solution are (long
Degree 5-100mm, 100 μm of -10mm of width) and apply the Parameter Conditions such as the electric-field intensity (3-300V/cm) of DC voltage to divide
From phycocyanin and bovine hemoglobin.Embodiment 2-10 separates the operational circumstances of phycocyanin and bovine hemoglobin specifically such as table 1
It is shown.
The embodiment 2-10 of table 1 separates phycocyanin and the operational circumstances of bovine hemoglobin.
As it can be seen from table 1 using isoelectric focusing and separation method described in embodiment 1, do not changing other specification bar
In the case of part, by the concentration (0.1mM-1M), the catholyte NH that adjust anodolyte HAc solution3·H2O solution
Concentration (0.1mM-1M), the length and width dimensions of paper passage (length 5-100mm, 100 μm of -10mm of width, large scale can first separation more
The both sexes sample of multiple types) and apply the parameters such as the electric-field intensity (3-300V/cm) of DC voltage, it can assemble and isolate
Phycocyanin and bovine hemoglobin, and the protein component for focusing on separation is easily recycled, while also it is beneficial to mass spectral characteristi.
The paper passage of the present invention can also use negative electrode electricity except anodolyte HAc solution can be used to be soaked
Solve matter NH3·H2O solution is soaked, the effect of wetting be in order that whole circuit formed can power circuit, in order in paper substrate
Focused on analytical equipment using isoelectric focusing method and isolate protein component.
The Anolyte solution of the present invention can also use volatility weak acid except using above-mentioned weak acid HAc solution
HCOOH solution, HF solution, H2S solution and H2CO3Solution, following acid solution can also be used certainly：Such as perchloric acid
HClO4, hydroiodic acid HI, sulfuric acid H2SO4, hydrobromic acid HBr, hydrochloric acid HCl, nitric acid HNO3, acid iodide HIO3, oxalic acid H2C2O4, sulfurous acid
H2SO3, phosphoric acid H3PO4, pyruvic acid CH3COCOOH, nitrous acid HNO2, citric acid C6H8O7, hydrofluoric acid HF, malic acid C4H6O5, Portugal
Grape saccharic acid C6H12O7, carbonic acid H2CO3, lactic acid CH3CH (OH) COOH, benzoic acid C6H5COOH, acrylic acid CH2=CHCOOH, propionic acid
CH3CH2COOH, stearic acid CH3(CH2)16COOH, hydrosulphuric acid H2S, hypochlorous acid HClO, boric acid H3BO3, silicic acid H2SiO3It is and acid
Buffer system etc..
The catholyte of the present invention is except using above-mentioned NH3·H2O solution, other alkaline solutions can also be substituted for,
Such as potassium hydroxide KOH, sodium hydroxide NaOH, barium hydroxide Ba (OH)2, calcium hydroxide Ca (OH)2And alkaline buffer system etc..
Further, both sexes components etc. are not carried out by carrier ampholyte in the opening paper-based devices shown in Fig. 1
Electrofocusing and the method for separation, by adjusting the concentration (i.e. pH value) of acid and alkaline electrolyte, formed on paper passage corresponding
PH scopes, isoelectric point can focus in the amphiphatic molecule of this pH scope.As shown in Figure 6 a, anode supporting electrolyte is 10mM
Containing the HEC that mass body fraction is 0.2% and paper passage is soaked with it in HAc, pH=4.3, HAc solution；Negative electrode is 0.5mg
mL-1Cromoci (pI-10.0) protein sample, sample substrate are 1M NH3·H2O solution, pH=11.8, other experiment conditions
Referring to embodiment 1.Understand, cromoci is focused on the paper passage that pH scopes are 4.3-11.8.Thus, anode selects
With acid supporting electrolyte, negative electrode selects alkaline supporting electrolyte, you can certain pH scopes, isoelectric point are formed on paper passage
It can obtain focusing on separation in the amphiphatic molecule of this pH scope.
We with being verified as follows, and as shown in Figure 6 b, anode supporting electrolyte is 1mM H3PO4Solution, pH=
3.0, H3PO4Containing mass body fraction it is 0.2%HEC in solution, and with its wetting paper passage；Cathode pool：0.5mg mL-1Algae
Azurin and 1.0mg mL-1Bovine hemoglobin, sample substrate are 1mM NaOH, pH=9.4, and other experiment conditions are referring to embodiment
1.Understand, when anode and cathode-supported electrolyte select phosphoric acid and sodium hydroxide respectively, phycocyanin and bovine hemoglobin also obtain
Focusing separation is arrived.
When from volatility weak acid solution and/or exemplary volatile weak base solution, mass spectrum is friendly, so as to be advantageous to separate and recover
The mass spectral characteristi of component, when separation component carries out mass spectral characteristi, it can handle without desalination operation, directly join with mass spectral analysis
With so as to reduce operational sequence.
The anodolyte of the present invention is preferably volatility weak acid Solution H Ac solution, and catholyte is preferably volatility
Weak base NH3·H2O solution.The benefit of selective volatilization weak acid and weak base is：On the one hand, H can be provided respectively+And OH-；The opposing party
Face, HAc and NH3·H2O is volatile component, is mass spectrum friendly supporting electrolyte, is advantageous to recovery and the matter of separation component
Stave is levied.When separation component carries out mass spectral characteristi, it can handle, be directly combined with mass spectral analysis, so as to drop without desalination operation
Low operational sequence, will not also introduce new material, cause interference during to other follow-up analysis (such as mass spectral analysis) combinations.
In a word, the present invention is without a range of freedom or immobilization carrier ampholytes, in paper substrate analytical equipment
On realized using the method for carrier-free ampholytes isoelectric focusing complicated both sexes sample (protein sample) quick focusing and
Separation, not only reduces cost, and avoids carrier ampholyte and be combined with other follow-up analyses (such as mass spectral analysis)
Caused interference.
In the present invention, both sexes sample is loaded directly into in acid anodic dissolution or alkaline cathode solution or on paper passage,
With both sides advantage：On the one hand both sexes sample can be made powered, after DC voltage is applied, is advantageous to both sexes sample component
Migrated in the presence of electric field；Another aspect simplified operation.
The technical principle of the present invention is described above in association with embodiment.These descriptions are intended merely to explain the present invention
Principle, and limiting the scope of the invention can not be construed in any way.Based on explanation herein, art technology
Personnel, which need not pay creative work, can associate other embodiments of the present invention, and these modes fall within this
Within invention protection domain.
Claims (7)
1. a kind of isoelectric focusing of carrier-free ampholytes and the method for separation, it is characterised in that methods described is led to using paper
Road, acid solution, alkaline solution, both sexes sample, two electrodes and dc source composition loop, described two electrodes insert respectively
In acid solution and alkaline solution, the paper passage is using its both ends point after the wetting of any of acid solution and alkaline solution
Do not contacted with acid solution and alkaline solution, described two electrodes are connected by wire with dc source, and both sexes sample directly adds
It is loaded onto in acid solution or alkaline solution or on paper passage.
2. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 1 and the method for separation, it is characterised in that
The concentration of the acid solution is 0.1mM-1.0M, and the concentration of the alkaline solution is 0.1mM-1.0M.
3. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 2 and the method for separation, it is characterised in that
The acid solution is volatility weak acid HAc solution or HCOOH solution, and the alkaline solution is exemplary volatile weak base NH3·H2O is molten
Liquid.
4. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 3 and the method for separation, it is characterised in that
Anode in described two electrodes uses platinum electrode, and negative electrode uses platinum electrode.
5. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 4 and the method for separation, it is characterised in that
The paper passage is the split tunnel being made using Fiber Materials.
6. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 5 and the method for separation, it is characterised in that
The paper passage is glass fibre membrane, and the length of the paper passage is 5-100mm, and width is 100 μm of -10mm.
7. a kind of isoelectric focusing of carrier-free ampholytes as claimed in claim 6 and the method for separation, it is characterised in that
The electric-field intensity scope put on the paper passage is 3-300V/cm.
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| CN101004384A (en) * | 2006-12-22 | 2007-07-25 | 吉林大学 | Raman spectrum method for detecting surface reinforcement of protein group |
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| CN107488208B (en) | 2020-06-05 |
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