CN109270153A - A kind of no ampholytes free flow isoelectric focusing electrophoresis separation method - Google Patents
A kind of no ampholytes free flow isoelectric focusing electrophoresis separation method Download PDFInfo
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- CN109270153A CN109270153A CN201811429548.2A CN201811429548A CN109270153A CN 109270153 A CN109270153 A CN 109270153A CN 201811429548 A CN201811429548 A CN 201811429548A CN 109270153 A CN109270153 A CN 109270153A
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Abstract
This application discloses a kind of no ampholytes free flow isoelectric focusing electrophoresis separation methods, step 1: being pumped into the disengagement chamber of free stream cataphoresis device using transfer tube by mobile phase is run, and keep mobile phase stable operation；Step 2: the anode electrode liquid pump in electrode liquid storage tank is entered in the cathode chamber of free stream cataphoresis device using electrode solution circulating pump and electrode solution is kept to recycle, the negative electrode liquid pump in electrode liquid storage tank is entered in the anode chamber of free stream cataphoresis device and electrode solution is kept to recycle；Step 3: applying voltage, adjusting voltage to appropriate value, and holding meanss stable operation in two electrodes, it is made to automatically form stable pH gradient between the rear portion of disengagement chamber, electrode.The advantage of the application is: method is simple and easy, and required reagent price is cheap, will not have an impact to sample detection after separation.
Description
Technical field
The present invention relates to free stream cataphoresis separation technology fields, are a kind of no ampholytes free flows etc. specifically
Electric focusing separation method.
Background technique
Protein is the material base of vital movement, many kinds of, vdiverse in function.It is to be connected by amino acid by amido bond
Large biological molecule being connected into, with specific structure and function.It is able to carry out various biological functions, makes in DNA
The information contained is shown.Protein is the most direct agent of vital movement, and the only research of protein group could be from root
Life process is illustrated on this.And the isolation technics of protein is the basis of proteome research.But since protein itself is had
Some special natures are difficult to use conventional isolation technics and are efficiently separated.
Free stream cataphoresis is a kind of continuous full liquid phase separation techniques, and device is formed by two pieces apart from close parallel-plate
One thin disengagement chamber.Operation mobile phase is flowed through from disengagement chamber, forms stable laminar flow.Sample enters separation by injection port
After chamber, outlet end is flowed to by liquid stream driving.An orthogonal cross is loaded by the liquid laminar flow to longitudinal flow in device simultaneously
To electric field, make the electrically charged sample in laminar flow that different degrees of cross occur according to different electrophoretic mobilities under the action of electric field force
To offset, to achieve the effect that separation.Sample continuously enters in free stream cataphoresis separation process, no solid support matrix, and
Isolating environment is mild, prepared by the separation especially suitable for biological samples such as protein.Cao had developed at happiness seminar in 2013
A kind of easy-operating stable free stream cataphoresis device with cooling system, greatly improves grasping for free stream cataphoresis device
The property made.The advantages of free stream cataphoresis can combine with other electrophoretic techniques, make full use of other electrophoretic techniques, so that free flow
Electrophoresis can play a role in more fields.Most common several clastotypes are free flow zone electrophoresis, free flow respectively
Isoelectric focusing, free flow isotachophoresis, free flow electric-force gradient electrophoresis etc..Cao developed a kind of shifting in 2017 at happiness seminar
Dynamic reaction interface free stream cataphoresis method, the continuous concentration for protein.Free flow isoelectric focusing electrophoresis is in common freedom
There is highest resolution ratio, so being typically used to the separation of more difficult separation sample in flow electrophoresis clastotype.Cao is at happiness project
Group was successfully prepared endogenous mitochondrial using circulation free flow isoelectric focusing device in 2017.This method uses a series of
High concentration ampholytes with pH gradient establishes pH in circulation free flow isoelectric focusing device as operation mobile phase
Gradient separates sample.Isoelectric focusing is a kind of hand for being mainly used for Separation of Proteins occurred the 1960s
Section.Due to its resolution ratio with higher, so being rapidly progressed.Its basic principle is to utilize various protein examples
Isoelectric point it is different, the separation that protein is carried out in a continuous linear pH gradient is analyzed.The process of isoelectric focusing is one
The process that a sample focuses, therefore its band diffusion problem is smaller, can achieve the purpose that separation and concentration simultaneously.It is traditional from
During stream isoelectric focusing electrophoresis, specific ampholytes buffer solution is needed to establish pH gradient.But ampholytes
Solution is not only expensive, thereby increases and it is possible to can generate interaction with sample segment, influence the sensitivity of ultraviolet detection.Group after separation
When point needing to connect Mass Spectrometer Method, it is necessary to be removed these ampholytes by complicated means.Therefore it is electrolysed without using both sexes
Matter buffer solution, which carries out free flow isoelectric focusing electrophoresis, will be a kind of ideal operating method.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, this application provides a kind of no ampholytes free flow isoelectric focusing
Electrophoresis separating method.
The application the technical solution adopted is that:
A kind of no ampholytes free flow isoelectric focusing electrophoresis separation method, the specific steps are that:
Step 1: being pumped into the disengagement chamber of free stream cataphoresis device using transfer tube by mobile phase is run, and keep flowing
Phase stable operation；
Step 2: the anode electrode liquid pump in electrode liquid storage tank is entered free stream cataphoresis device using electrode solution circulating pump
In cathode chamber and electrode solution is kept to recycle, the negative electrode liquid pump in electrode liquid storage tank is entered to the anode chamber of free stream cataphoresis device
In and keep electrode solution to recycle；
Step 3: apply voltages in two electrodes, adjust voltage to appropriate value, and holding meanss stable operation, make its
The rear portion of disengagement chamber automatically forms stable pH gradient between electrode.
Above-mentioned free flow isoelectric focusing electrophoresis method use a kind of free stream cataphoresis device, the free stream cataphoresis device its
Comprising running mobile phase liquid storage bottle, mobile phase transfer tube is run, gas-liquid buffer unit, free stream cataphoresis chamber, anode electrode room is born
Pole electrode chamber, anode electrode liquid circulating pump, negative electrode liquid circulating pump, anode electrode liquid liquid storage bottle, negative electrode liquid liquid storage bottle,
High voltage power supply, component collection device；Operation mobile phase liquid storage bottle is attached by hose and operation mobile phase transfer tube, is run
Mobile phase transfer tube is attached by hose and gas-liquid buffer unit, and gas-liquid buffer unit passes through hose and free stream cataphoresis chamber
It is attached, free stream cataphoresis chamber is attached by hose and component collection device, and free stream cataphoresis chamber is handed over by cation
Film is changed to be attached with cathode chamber, cathode chamber is attached by electrode bar and high-voltage power cathode, cathode chamber by hose with
Anode electrode liquid circulating pump is attached, and anode electrode liquid circulating pump is attached by hose and anode electrode liquid liquid storage bottle,
Free stream cataphoresis chamber is attached by anion-exchange membrane and anode chamber, anode chamber by electrode bar and high voltage power supply cathode into
Row connection, anode chamber are attached by hose and negative electrode liquid circulating pump, and negative electrode liquid circulating pump is by hose and bears
Pole electrode solution liquid storage bottle is attached.
For free stream cataphoresis device Anodic using acid electrode solution, the concentration of the acidity electrode solution is 0.01mol/L-
1mol/L。
For cathode using alkaline electrode solution, the concentration of the alkalinity electrode solution is 0.01mol/L- in free stream cataphoresis device
1mol/L。
Free stream cataphoresis device uses buffer solution as operation mobile phase, the buffer concentration in the process of running
For 0.001mol/L-0.1mol/L.
It is 1ml/min-100ml/min that free stream cataphoresis device, which runs flow rate of mobile phase,.
Free flow isoelectric focusing electrophoresis method can form pH2-10, the preferably pH gradient of 3-8 under electric field force effect.
This application provides application of no ampholytes free flow isoelectric focusing electrophoresis in Separation of Proteins.
Compared with prior art, the beneficial effects of the present invention are:
PH gradient is formed as operation mobile phase without using expensive special ampholytes.Electrode solution in electrode chamber
In hydrogen ion and hydroxide ion can automatically form pH gradient free stream cataphoresis is intracavitary under the action of electric field force.
The indoor anode electrode liquid of anode electrode passes through in anode electrode liquid circulating pump and anode electrode liquid liquid storage bottle just
Pole electrode solution is recycled, and the indoor anode electrode liquid of anode electrode is made to keep the stabilization of concentration.The indoor cathode of negative electrode
Electrode solution is recycled by the negative electrode liquid in negative electrode liquid circulating pump and negative electrode liquid liquid storage bottle, makes negative electrode
Indoor negative electrode liquid keeps the stabilization of concentration.To ensure that the intracavitary pH gradient of free stream cataphoresis is stablized.
Used buffer solution will not interact with sample, improve relative to ampholytes as operation mobile phase
Sensitivity when UV detection.
When component after free stream cataphoresis separation needs to connect Mass Spectrometer Method, very due to the buffer concentration that uses
It is low, it not will increase ionic current and without desalting processing.
Detailed description of the invention
Fig. 1 is free stream cataphoresis schematic device.
Fig. 2 is free stream cataphoresis disengagement chamber schematic diagram.
Fig. 3 is pH gradient curve；
Fig. 4 is after egg white powder separation component is detected by Matrix Assisted Laser Desorption ionization source time of-flight mass spectrometer
Mass spectrogram；Fig. 4 a is the schematic diagram in the first hole, and Fig. 4 b is the schematic diagram in the 4th hole, and Fig. 4 c is the schematic diagram in the 9th hole.
The label in accompanying drawing is: 1-operation mobile phase transfer tube, 2-negative electrode solution circulating pumps, 3-anode electrode liquid circulation
Pump, 4-sampling pumps, 5-negative electrode liquid liquid storage bottles, 6-operation mobile phase liquid storage bottles, 7-sample solution liquid storage bottles, 8-just
Pole electrode solution liquid storage bottle, 9-high voltage power supplies, 10-inflators, 11-gas-liquid buffer units, 12-free stream cataphoresis chambers, 13-groups
Divide collection device；121-anode electrode rooms, 122-negative electricity pole rooms, 123-free stream cataphoresis disengagement chambers, 124- operation flowing
Phase input channel.
Specific embodiment
Detailed technology content for the present invention and its preferably implementation are as follows, but can not limit the content of present invention, cooperate
Accompanying drawing content is described as follows.
Embodiment 1
Refering to Figure 1, free stream cataphoresis schematic device used in this application.This device includes: an operation stream
Dynamic phase transfer tube (1) is used to that the operation mobile phase in operation mobile phase liquid storage bottle (6) to be driven to enter in gas-liquid buffer unit (11).
The bubble removal in mobile phase will be run using the inflator (10) in gas-liquid buffer unit (11), makes to run mobile phase full of freely
Flow electrophoresis cavity (12)；Negative electrode liquid circulating pump (2) is for driving the negative electrode liquid in negative electrode liquid fluid reservoir (5) to follow
Ring, anode electrode liquid circulating pump (3) is for driving the anode electrode liquid in anode electrode liquid fluid reservoir (8) to recycle；One sampling pump
(4) for the sample in sample solution liquid storage bottle (7) to be pumped into free stream cataphoresis cavity (12)；One high voltage power supply (9) is for mentioning
For device voltage；One component collection device (13).
As shown in fig.2, free stream cataphoresis chamber schematic diagram in free stream cataphoresis device used in the present invention.This device packet
It includes: operation mobile phase input channel (124)；One anode electrode room (121) is separated by cation-exchange membrane with free stream cataphoresis
Chamber (123) is separated by；One negative electricity pole room (122) is separated by by anion-exchange membrane and free stream cataphoresis disengagement chamber (123).
During free flow isoelectric focusing electrophoresis, on the direction of operation mobile phase operation, mainly flowed by operation
Phase transfer tube (1) provides motive force, makes to run mobile phase and sample solution together by the entrance flow direction of free stream cataphoresis chamber (12)
Outlet.The process that can regard an isoelectric focusing on perpendicular direction as is flowed with operation.High voltage power supply in this process
(9) provide voltage, make the hydrogen ion in anode electrode room (121) under electric field action by anode electrode room (121) sun from
Proton exchange is mobile to cathode, and the hydroxide ion in negative electricity pole room (122) passes through negative electricity pole room under electric field action
(122) anion-exchange membrane is mobile to anode.Operation mobile phase near anode is in since the increase of hydrogen ion concentration makes it
Acidity, and the operation mobile phase near cathode is since hydroxide ion concentration increase is in alkalinity.In free stream cataphoresis chamber (12)
Middle section is formed because of the migration of hydrogen ion and hydroxide ion under electric field action close to linear pH value gradient, pH gradient
Curve is as shown in Figure 3.
Embodiment 2
The first step opens operation mobile phase transfer tube (1) and is pumped into the operation mobile phase run in mobile phase liquid storage bottle (6)
In gas-liquid buffer unit (11).The bubble removal in mobile phase will be run using the inflator (10) in gas-liquid buffer unit (11),
Make to run mobile phase full of free stream cataphoresis cavity (12) and by flow control in 1ml/min.Open negative electrode liquid circulating pump
(2) and anode electrode liquid circulating pump (3) keeps the positive and negative anodes in negative electrode liquid storage tank (5) and anode electrode liquid fluid reservoir (8) electric
Pole liquid pump enters in the positive and negative anodes electrode chamber of free stream cataphoresis chamber, and electrode solution is kept to recycle.It opens high voltage power supply (9), is adjusted to
400V voltage.The holding meanss stable 30min time, make its stabilization.Sampling pump (4) are opened by sample solution liquid storage bottle (7)
In egg white sample solution be pumped into free stream cataphoresis cavity (12) with the speed of 1ml/min.It transports under the action of electric field force
After row 1h, sample obtains separation concentration, is then collected by component collection device (13).Egg white powder separation component after collection
It is detected by Matrix Assisted Laser Desorption ionization source time of-flight mass spectrometer, obtained result is as shown in Figure 4: by using this
Free flow isoelectric focusing electrophoresis method separates three kinds of protein components in egg clear solution fully according to its isoelectric point.And
Desalting processing is not necessarily to before Mass Spectrometer Method.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, without departing from the inventive concept of the premise, can also make several improvements and modifications, these improvements and modifications also should be regarded as
In protection scope of the present invention.
Claims (8)
1. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method, which is characterized in that the specific steps are that:
Step 1: being pumped into the disengagement chamber of free stream cataphoresis device using transfer tube by mobile phase is run, and keep mobile phase steady
Fixed operation；
Step 2: the anode electrode liquid pump in electrode liquid storage tank to be entered to the anode of free stream cataphoresis device using electrode solution circulating pump
In room and electrode solution is kept to recycle, the negative electrode liquid pump in electrode liquid storage tank is entered in the anode chamber of free stream cataphoresis device simultaneously
Keep electrode solution circulation；
Step 3: applying voltage, adjusting voltage to appropriate value, and holding meanss stable operation in two electrodes, separating it
The rear portion of chamber automatically forms stable pH gradient between electrode.
2. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
It is buffer solution in, the operation mobile phase, wherein salinity is 0.001mol/L-0.1mol/L, pH value 2-12, preferably
4-10,.
3. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
In the anode electrode liquid is acid solution, pH value range 2-7, concentration 0.01mol/L-1mol/L.
4. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
In the negative electrode liquid is alkaline solution, pH value range 7-12, concentration 0.01mol/L-1mol/L.
5. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
In the rear portion of the disengagement chamber refers to the position of the 50%-70% of disengagement chamber longitudinal direction.PH gradient range is 2-10, preferably 3-
9。
6. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
In the pH gradient automatically formed between electrode is the transverse direction along disengagement chamber, and the operation mobile phase near anode is dense due to hydrogen ion
The increase of degree makes it in acidity, and the operation mobile phase near cathode is since hydroxide ion concentration increase is in alkalinity, its it
Between, because of the migration of hydrogen ion and hydroxide ion under electric field action, formed close to linear pH value gradient.
7. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1 uses freely
Flow electrophoresis device, which is characterized in that the free stream cataphoresis device, comprising operation mobile phase liquid storage bottle, operation mobile phase is driven
Dynamic pump, gas-liquid buffer unit, free stream cataphoresis chamber, anode electrode room, negative electricity pole room, anode electrode liquid circulating pump, negative electricity
Pole liquid circulating pump, anode electrode liquid liquid storage bottle, negative electrode liquid liquid storage bottle, high voltage power supply, component collection device；Run mobile phase
Liquid storage bottle is attached by hose and operation mobile phase transfer tube, and operation mobile phase transfer tube is filled by hose and gas-liquid buffering
Set and be attached, gas-liquid buffer unit is attached by hose and free stream cataphoresis chamber, free stream cataphoresis chamber by hose with
Component collection device is attached, and free stream cataphoresis chamber is attached by cation-exchange membrane and cathode chamber, and cathode chamber passes through
Electrode bar is attached with high-voltage power cathode, and cathode chamber is attached by hose and anode electrode liquid circulating pump, anode electricity
Pole liquid circulating pump is attached by hose and anode electrode liquid liquid storage bottle, and free stream cataphoresis chamber is by anion-exchange membrane and bears
Pole room is attached, and anode chamber is attached by electrode bar and high voltage power supply cathode, and anode chamber passes through hose and negative electrode
Liquid circulating pump is attached, and negative electrode liquid circulating pump is attached by hose and negative electrode liquid liquid storage bottle.
8. a kind of no ampholytes free flow isoelectric focusing electrophoresis separation method according to claim 1, feature exist
In can be used for the preparative separation of protein.
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| CN201811429548.2A CN109270153B (en) | 2018-11-28 | 2018-11-28 | Non-amphoteric electrolyte free-flow isoelectric focusing electrophoresis separation method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109759150A (en) * | 2019-01-30 | 2019-05-17 | 中山大学 | Controllable extraining sampling device, sample injection method and application based on micro- free stream cataphoresis |
| CN111560063A (en) * | 2020-05-12 | 2020-08-21 | 蚌埠医学院 | Raw material medicine purification device for animal pancreas-derived insulin and use method |
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| US20110206725A1 (en) * | 2008-09-05 | 2011-08-25 | Immunobiology Limited | Method for purifying protein complexes |
| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
| CN104307367A (en) * | 2014-10-27 | 2015-01-28 | 上海交通大学 | Reciprocating type free flow isoelectric focusing electrophoresis device and method |
-
2018
- 2018-11-28 CN CN201811429548.2A patent/CN109270153B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110206725A1 (en) * | 2008-09-05 | 2011-08-25 | Immunobiology Limited | Method for purifying protein complexes |
| CN104056468A (en) * | 2014-06-25 | 2014-09-24 | 上海交通大学 | Electrofocusing method for separating amphoteric matter without amphoteric electrolyte |
| CN104307367A (en) * | 2014-10-27 | 2015-01-28 | 上海交通大学 | Reciprocating type free flow isoelectric focusing electrophoresis device and method |
Non-Patent Citations (1)
| Title |
|---|
| SONG-FANG XIE ET.AL: "Carrier ampholyte-free isoelectric focusing on a paper-based analytical device for the fractionation of proteins", 《JOURNAL OF SEPARATION SCIENCE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109759150A (en) * | 2019-01-30 | 2019-05-17 | 中山大学 | Controllable extraining sampling device, sample injection method and application based on micro- free stream cataphoresis |
| CN111560063A (en) * | 2020-05-12 | 2020-08-21 | 蚌埠医学院 | Raw material medicine purification device for animal pancreas-derived insulin and use method |
| CN111560063B (en) * | 2020-05-12 | 2022-11-01 | 蚌埠医学院 | Raw material medicine purification device for animal pancreas-derived insulin and use method |
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