CN106770595B - Electrophoresis tank for Protein Separation and the column gel electrophoresis apparatus using it - Google Patents
Electrophoresis tank for Protein Separation and the column gel electrophoresis apparatus using it Download PDFInfo
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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
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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
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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Abstract
一种用于蛋白分离的电泳槽及采用其的柱状凝胶电泳装置，该电泳槽包括正极侧的缓冲溶液槽、负极侧的缓冲溶液槽、位于中间的冷却水槽和位于正极侧的洗脱溶液槽；所述电泳槽中设置有安放柱状凝胶管的位置。所述电泳槽通过3D打印技术制造，例如采用高分子聚合物材料制成，优选由PLA塑料制成。本发明的电泳槽及采用其的柱状凝胶电泳装置根据所填充的聚丙烯酰胺的不同以及结合ICP‑MS等仪器可用于不同分子量蛋白的离线分离和在线分离及检测，且具有分析时间短，成本低，制造过程简单，死体积小，分离条件选择范围广，分离度高，可根据实际需要进行改装等特点，并具有不断优化的可能。
An electrophoresis tank for protein separation and a columnar gel electrophoresis device using the same, the electrophoresis tank includes a buffer solution tank on the positive side, a buffer solution tank on the negative side, a cooling water tank in the middle, and an elution solution on the positive side The electrophoresis tank is provided with a position for placing a columnar gel tube. The electrophoresis tank is manufactured by 3D printing technology, for example, it is made of high molecular polymer material, preferably made of PLA plastic. The electrophoresis tank of the present invention and the columnar gel electrophoresis device using the same can be used for off-line separation, on-line separation and detection of proteins of different molecular weights according to the difference of the filled polyacrylamide and in combination with ICP-MS and other instruments, and has the advantages of short analysis time, Low cost, simple manufacturing process, small dead volume, wide selection of separation conditions, high separation degree, can be modified according to actual needs, etc., and has the possibility of continuous optimization.
Description
Technical field
The invention belongs to biochemistries to test and analyze technical field, relate more specifically to a kind of electrophoresis for Protein Separation
Slot and the column gel electrophoresis apparatus for using it.
Background technique
Basic component of the protein as organism, is the executor of the various critical functions of organism, to protein
The research of composition, structure and function has important research significance and real value.Wherein electrophoretic techniques is that Separation of Proteins is surveyed
Most widely used and most efficient method in fixed.
Electrophoretic techniques mainly includes isoelectric focusing electrophoresis (IEF) and polyacrylamide gel electrophoresis (PAGE), PAGE be with
Polyacrylamide gel with netted stereochemical structure is supporting dielectric, under electric field action, protein molecule root to be separated
The apparent zone of many items is separated into according to molecular charge amount, size and shape difference.PAGE can be divided into denaturation (SDS- again
PAGE) with native gel electrophoresis (Native-PAGE).Denaturing gel electrophoresis (SDS-PAGE) passes through dodecyl sodium sulfate
(SDS) it is reacted with albumen and makes protein inactivation, protein can be converted to molecular weight is different and the similar substance of structure, therefore nothing
By albumen property how, can be separated according to the difference of molecular weight, the small albumen of mass-to-charge ratio is fastly, far mobile, matter
Lotus is more mobile than big albumen slowly.SDS-PAGE is easy to operate flexibly, and high resolution, favorable reproducibility is the most widely used
Protein stripping technique.
Different according to the device form of support, gel electrophoresis can be divided into flat electrophoresis, vertical board-like electrophoresis and column again
Shape electrophoresis.Wherein column gel is more advantageous to the concentration and the collection after separation and the combination with subsequent detection system of sample.
Column gel electrophoresis can select the glass tube of different inner diameters and length according to demand, and filling agarose can be used for point in glass tube
Freestone acid, filled polypropylene acrylamide gel can be used for protein isolate matter.Protein after polyacrylamide electrophoresis separates passes through elution
Device is eluted, and is analyzed and detected with the combination of icp ms (ICP-MS) technology.Column is solidifying
Gel electrophoresis has been successfully applied to separate DNA, RNA and small-molecule substance online with the combination of ICP-MS technology.
However although to can be used for polypeptide, RNA, DNA and albumen etc. more for existing commercialization column gel electrophoresis apparatus
The separation and detection of kind substance, universality is good, but also causes the internal diameter of its gel column larger, and washing device dead volume is larger, because
This entire polyacrylamide electrophoresis device is all lower to the separative efficiency and sensitivity for analysis of albumen, and cost is high, operates more complex.
How polyacrylamide electrophoresis device is optimized and improvement is an important factor for influence protein analysis is with detection.
3D printing technique is a kind of technology completely contradicted with traditional material processing method, which is based on three-dimensional CAD
Model data, come Fast Construction object by way of increasing material and successively manufacturing.It uses directly manufacture and corresponding mathematical modulo
The manufacturing method of the completely the same three dimensional physical physical model of type simultaneously realizes print procedure by digital technology file printing machine.
The characteristics of this technology, which is it almost, can produce the article of any shape, and eliminates traditional handicraft and multiple tracks is needed to process
The tedious procedure of program, the manufacturing cycle is short, at low cost.In recent years, advanced manufacture of the 3D printing technique as a frontier nature
Technology is grown rapidly, and gradually played an important role in the fields such as industry manufacture, biomedical, building manufacture, culture and arts.
But there has been no the precedents that 3D printing technique is applied to column gel electrophoresis apparatus at present.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of electrophoresis tank for Protein Separation and using its column
At least one of shape gel electrophoresis apparatus, to solve the above technical problems.
To achieve the above object, as one aspect of the present invention, the present invention provides a kind of electricity for Protein Separation
Swimming slot, which is characterized in that the buffer solution slot of buffer solution slot, negative side including side of the positive electrode is located in the middle cooling trough
With the elution solution tank for being located at side of the positive electrode；The position for placing column gel tube is provided in the electrophoresis tank.
Wherein, the electrophoresis tank is manufactured by 3D printing technique.
Wherein, the electrophoresis tank is made of macromolecule polymer material, is preferably made of PLA plastics.
Wherein, the depth of the electrophoresis tank is 3.5-5cm, width 8-12cm, total length 18-22cm.
Wherein, the length of cooling trough is 5.5-9cm in the electrophoresis tank；The buffer solution slot and elution solution of side of the positive electrode
The length of slot is 5-6cm；The length of the buffer solution slot of negative side is 5-6cm.
Wherein, the length of cooling trough is 5.5cm in the electrophoresis tank；The buffer solution slot and elution solution tank of side of the positive electrode
Length be 6cm；The length of the buffer solution slot of negative side is 5cm.
Wherein, the buffer solution slot of the side of the positive electrode wall adjacent with elution solution tank is equipped with elution pipe jack, in institute
The outside for stating the buffer solution slot of side of the positive electrode and buffer solution slot the same side of negative side is respectively arranged with the electrode of positive and negative electrode
Seat, and electrode is housed.
Wherein, the top outside the cooling trough side is provided with movable snap-gauge, for fixing column gel tube and envelope
Close cooling trough；Described activity snap-gauge one end and the other side of cooling trough are respectively set there are two tightening screw hole, when use
For being inserted into screw tightening activity snap-gauge.
As another aspect of the present invention, the present invention also provides a kind of column gel electrophoresis apparatus, which is characterized in that
The column gel electrophoresis apparatus uses electrophoresis tank as described above.
Wherein, the column gel electrophoresis apparatus is horizontal column gel electrophoresis apparatus, and column gel tube therein is horizontal
It places.
Based on above scheme it is found that horizontal column gel electrophoresis apparatus of the invention has the following beneficial effects: (1) this hair
Bright horizontal column gel electrophoresis apparatus can be used for the quick offline separation of albumen；(2) horizontal column gel electrophoresis of the invention
The analysis instruments such as device combination ICP-MS can realize that protein sample is fast and accurately separated and detected online, improve point of albumen
Sensitivity from efficiency and analysis；(3) difference of column gel tube of the invention according to the polyacrylamide gel of filling, cooperation
Electrophoresis tank uses the quick separating that different proteins can be achieved；(4) horizontal column gel electrophoresis apparatus main body of the invention uses
3D printing manufacture, material therefor is about 300 grams, and about 500 yuans of cost or so, less than 18 hours of entire manufacturing process,
Light weight, low in cost, manufacturing process is fast and simple；(5) washing device dead volume of the invention is small, it is cheap, it can be achieved that
Disposable or reuse；(6) column gel electrophoresis apparatus structure of the invention is simple, easily operated；(7) gel of the invention
Electrophoretic apparatus Protein Separation efficiency with higher, and with low in cost, manufacturing process is simple, dead volume is small, quick and precisely
The features such as, and there is the possibility for continuing optimization.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of column gel tube of the invention；
Fig. 2 is the structural schematic diagram of electrophoresis tank of the invention；
Fig. 3 A and Fig. 3 B are the sectional view of washing device left-half of the invention and the STRUCTURE DECOMPOSITION of washing device respectively
Schematic diagram；
Fig. 4 A and Fig. 4 B are using column gel electrophoresis Protein Separation device (column gel tube and electrophoresis of the invention respectively
Slot) Precision Plus Protein Dual Color protein standard substance produced to Bio-Rad company separated
As a result with the separating effect figure of SDS-PAGE provided by Bio-Rad company；
Fig. 5 is to two kinds using gel electrophoresis Protein Separation device combination ICP-MS of the invention with I127The standard of label
The electrophoresis result figure that albumen is separated and detected online, wherein peak 1 is RA albumen, and peak 2 is CA albumen.
Fig. 6 A is to three kinds using gel electrophoresis Protein Separation device combination ICP-MS of the invention with I127The standard of label
The electrophoresis result figure that albumen is separated and detected online, Fig. 6 B are using the commercialization electrophoretic apparatus by optimization in same batten
The result figure detected under part to same three kinds of standard proteins, wherein peak 1 is RA albumen, and peak 2 is CA albumen, and peak 3 is BSA
Albumen.
In upper figure, appended drawing reference meaning is as follows:
1.-- buffer solution slot；2.-- elutes solution tank；3.-- cooling trough；4.-- negative electrode；5.-- positive electrode；6.--
Wire electrode slot；7.-- gel tube placed hole；8.-- elutes pipe jack；The cooling water inlet 9.--；10.-- cooling water outlet；11.--
Movable snap-gauge；12.-- tightens screw hole；
21.-- gel tube jack；The small SPE sieve plate putting hole of 22.--；23.-- elution pipe；24.-- interface；25.--20μm
SPE sieve plate (small SPE sieve plate)；26.--3kD dialysis membrane；27.--3mm SPE sieve plate (big SPE sieve plate)；The big SPE sieve plate of 28.--
Putting hole；29.-- buffer solution intercommunicating pore.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in further detail.
The present invention relates to a kind of horizontal column gel electrophoresis apparatus and column gel therein for Protein Separation
Three main components such as pipe, electrophoresis tank and washing device.Wherein, electrophoresis groove body and washing device main body use 3D printing system
It makes, material is high molecular material, such as PLA plastics and synthetic resin, the case where having the advantages that according to actual sample,
Using the horizontal column gel electrophoresis apparatus and its column gel tube and electrophoresis tank that are included, it can be used for offline point of albumen
From；Column gel tube, electrophoresis tank and the washing device for being included with it using the horizontal column gel electrophoresis apparatus, in conjunction with
The analysis instruments such as ICP-MS can be used for the online separation and detection of albumen.
When specifically used, when needing offline inspection, different polyacrylamide gels is filled in column gel tube, to
Protein sample to be separated is added in column gel tube, and is placed in electrophoresis tank, then into the different slots of gel electrophoresis slot
It is separately added into buffer solution and cooling circulating water, fastening activity snap-gauge, and the electrode of electrophoresis tank is powered on, later albumen sample
Product can be separated offline in gel tube under the action of electric current, and whole process is easy to operate, and separative efficiency is high.
When needing on-line checking, different polyacrylamide gels is filled in column gel tube, by column gel tube
It is placed in electrophoresis tank, and is connect with washing device, the wherein side elution pipe of washing device passes through peristaltic pump and inductive coupling
Plasma mass spectrograph injection port connects, and protein sample to be separated is added in Xiang Zhuzhuan gel tube, and be placed in electrophoresis tank,
Then it is separately added into buffer solution into the different slots of gel electrophoresis slot, elutes solution and cooling circulating water, fastening activity snap-gauge,
And power on the electrode of electrophoresis tank, protein sample can be separated in gel tube under the action of electric current later, and with
Eluent enters ICP-MS under the action of peristaltic pump and is analyzed and detected.It is quickly quasi- to can be realized simultaneously protein sample in this way
True online separation and detection, and whole device is easy to operate, and dead volume is small, and separation and detection efficiency are high.
Specifically, horizontal column gel electrophoresis apparatus of the invention includes column gel tube, electrophoresis tank and selectable washes
De- device.
Wherein, which uses horizontal structure, i.e. column gel tube is horizontal positioned.Column gel
Different types of polyacrylamide gel can be filled in pipe, needed according to detection to select corresponding gel.For example, if desired dividing
From high-molecular-weight protein, then the gel of low concentration is filled；If desired separate low molecular amount albumen, then fill high concentration gel；If
It needs to separate wide molecular weight protein, then gradient gel can be filled.By validation trial and accounting, the length of column gel tube
It then can be preferably 4-6mm, further preferably 4mm, internal diameter according to gel filled how much settings, for example, 8-12cm, outer diameter
Preferably 2-2.2mm, further preferably 2.2mm, to significantly improve the separative efficiency for albumen.
Wherein, electrophoresis tank includes the buffer solution slot of positive and negative electrode side, is located in the middle cooling trough and is located at side of the positive electrode
Elution solution tank.The buffer solution slot of the side of the positive electrode wall adjacent with elution solution tank is equipped with elution pipe jack, slow at two
There is the electrode tip holder of positive and negative electrode in the outside for rushing solution tank side respectively, and electrode is housed, and the conducting Pt wire connecting with electrode passes through
Aperture on slot extends to two buffer solution trench bottoms along wire electrode card slot, in two buffer solution slots and cooling trough phase
There is gel tube placed hole on the wall connect respectively, the two sides of cooling trough are respectively equipped with cooling water inlet and cooling water outlet, cooling
Sink side outer tip end has movable snap-gauge, for immobilized gel pipe and closing cooling trough, movable snap-gauge one end and cooling water
The slot other side is respectively there are two tightening screw hole, for being inserted into screw tightening activity snap-gauge when use.
Preferably, electrophoresis tank uses macromolecule polymer material, such as PLA plastics to be made.It is further preferred that electrophoresis
Slot is manufactured by 3D printing technique.The depth of electrophoresis tank is, for example, 3.5-5cm, and width is, for example, 8-12cm, and total length is, for example,
18-22cm。
Wherein, the length of cooling trough is, for example, 6-9cm, side of the positive electrode buffer solution slot and elution solution tank in electrophoresis tank
Length is, for example, 5-6cm, and the length of negative side buffer solution slot is, for example, 5-6cm.
Preferably, the diameter of the gel tube placed hole on buffer solution cell wall in electrophoresis tank is, for example, 4mm.
Wherein, with life between the movable snap-gauge with corresponding electrophoresis cell wall of the cooling trough side outer tip end in electrophoresis tank
Material strip winding is tightened when use with two of screw insertion activity snap-gauge one end and the cooling trough other side respectively to avoid leak
To tighten movable snap-gauge in screw hole.
Washing device includes former and later two combinable parts, and the front end of first half is provided with long 6-8mm, diameter is
The jack of the column gel tube of 5.5-7.5mm, centre guarantee air-tightness added with rubber hose, and the centre of first half is length
1.3mm, the circular hole that diameter is 4mm, for placing agilent company 20 μm of SPE sieve plates produced (small SPE sieve plate), in circle
The two sides in hole pass through an elution pipe connection respectively, and the rear end of first half is provided with the interface of long 4.1mm, diameter 11.2mm
Connect with latter half.The circular hole that the front end of latter half is provided with long 1.6mm, diameter is 9.2mm, for placing Ai Jieer public affairs
3mm SPE sieve plate produced (big SPE sieve plate) is taken charge of, is placed with the production of MYM Bioisystech Co., Ltd in big SPE sieve plate front end
3kD dialysis membrane, the connecting hole communicated with buffer solution storage container is provided in the rear end of latter half.
Preferably, the main part of washing device is made for example, by using macromolecule polymer material, such as synthetic resin.Into
Preferably, washing device main part is manufactured one step by 3D printing technique.
In a preferred embodiment, gel tube of the invention when in use, is inserted into washing device by washing device
First half simultaneously contacts small SPE sieve plate.In another preferred embodiment, washing device is when in use by former and later two parts
It is bolted together, and tightly wraps interface with sealed membrane.
Preferably, washing device is using preceding needing sufficiently to soak large and small SPE sieve plate and dialysis membrane, and exclude air
To guarantee effect.
Preferably, two elution pipes are located at upper and lower ends to washing device when in use, the elution pipe of upper end passes through
Elution pipe jack on electrophoresis tank is inserted into elution solution tank, the casing connection of the elution pipe and wriggling pump side of lower end, and
Icp ms are introduced under the flow control of peristaltic pump.
When the horizontal column gel electrophoresis apparatus is used for the offline separation of albumen, need to simultaneously using column gel tube and
Two components of electrophoresis tank；When the horizontal column gel electrophoresis apparatus is used for the online separation of albumen, need solidifying using column simultaneously
Three sebific duct, electrophoresis tank and washing device components.
The invention also discloses a kind of gel electrophoresis albumen point using horizontal column gel electrophoresis apparatus as described above
From detection system.
As a preferred embodiment, the invention discloses a kind of horizontal column gel electrophoresis dresses for Protein Separation
It sets, which includes three column gel tube, electrophoresis tank and washing device components, and wherein column gel tube is added using quartz glass
Work is made, and electrophoresis groove body and washing device main body are printed using commercialization 3D printer.Electrophoresis groove body uses 3D
The CubePro3D desktop grade printer printing produced of Systems company, printed material are PLA plastics, consumptive material about 300g, consumption
When about 18 hours.Washing device main body is printed using Formlabs company Form2 desktop grade 3D printer produced, printing
Material is synthetic resin, consumptive material about 5g, time-consuming about 1 hour.
Another preferred embodiment of the present invention is described further with reference to the accompanying drawing.
As shown in Figure 1, the column gel length of tube for being used for Protein Separation is 8cm, outer diameter 4mm, internal diameter 2.2mm.
The column gel tube at work, needs inside to fill the polyacrylamide gel of different volumes and concentration, it
It is placed on the gel tube placed hole on electrophoresis tank of the invention, makes in conjunction with electrophoresis tank and washing device etc. of the invention afterwards
With after protein sample is added and is powered, protein sample can be mobile from cathode to anode under the action of electric current and be divided
From.
As shown in Fig. 2, the column gel electrophoresis slot for being used for Protein Separation is manufactured using PLA plastics, full weight is about 300
Gram, the electrophoresis groove depth is 4cm, width 10cm, total length 20cm.The electrophoresis tank includes: that two of positive and negative electrode side are slow
Rush solution tank 1, elution solution tank 2, cooling trough 3, negative electrode 4, positive electrode 5, wire electrode slot 6, gel tube placed hole 7, elution
Pipe jack 8, cooling water outlet 10, movable snap-gauge 11, tightens screw hole 12 at cooling water inlet 9.Electrophoresis tank includes four slots, point
It is not the two buffer solution slots 1, the cooling trough 3 of centre and the elution solution tank 2 of side of the positive electrode of positive and negative electrode side, wherein cold
But the length of sink 3 is 5.5cm, and the buffer solution slot 1 of side of the positive electrode and the length of elution solution tank 2 are 6cm, the buffering of negative side
The length of solution tank 1 is 5cm.There is positive and negative electrode 4,5 respectively in the outside of two buffer solution slot sides, positive and negative electrode side is delayed
Rush the side adjacent with cooling trough 3 of solution tank 1 has gel tube placed hole 7, a diameter of 4mm, with negative electrode 4 and positive electricity respectively
The conducting Pt wire that pole 5 connects is laid on the bottom of the buffer solution slot 1 of positive and negative anodes side along wire electrode slot 6 respectively, and side of the positive electrode delays
It rushes the wall adjacent with solution tank 2 is eluted of solution tank 1 and is equipped with elution pipe jack 8.The two sides of cooling trough 3 are equipped with cooling water inlet
9 and cooling water outlet 10, cooling trough side have movable snap-gauge 11, for immobilized gel pipe and closing cooling trough 3, active card
11 one end of plate and 3 other side of cooling trough are respectively there are two screw hole 12 is tightened, for tightening movable snap-gauge 11.
The electrophoresis tank at work, is separately added into the slow of 150ml or so in two buffer solution slots 1 of positive and negative electrode side
Solution is rushed, by long 8cm, outer diameter is that the column gel tube of 4mm is placed in gel tube placed hole 7, then detains movable snap-gauge 11
On, it is fixed by tightening screw hole 12 with two screws, wherein being wrapped with raw material band between movable snap-gauge and electrophoresis tank to avoid leakage
Negative electrode 4 and positive electrode 5 are connected on power supply by liquid by electric lead respectively, and appropriate elution solution is added in elution solution tank 2,
The washing device one end being used cooperatively.
As shown in Fig. 3 A, 3B, which includes former and later two combinable parts, first half
The jack 21 for the column gel tube that the front end divided is provided with a length of 8mm, diameter is 5.6mm, centre is added with rubber hose to guarantee
Air-tightness, the centre of first half is the circular hole 22 of long 1.3mm, diameter 4mm, for placing 20 μm produced of agilent company
SPE sieve plate (small SPE sieve plate) 25 has an elution pipe connection in the two sides of circular hole respectively, is provided in the rear end of first half
A length of 4.1mm, the interface 24 that diameter is 11.2mm connect with latter half.The front end of latter half is provided with long 1.6mm, diameter
For the circular hole 28 of 9.2mm, for placing Ai Jieer company 3mm SPE sieve plate produced (big SPE sieve plate) 27, before big sieve plate
End is placed with the 3kD dialysis membrane 26 of MYM Bioisystech Co., Ltd production, is provided with and buffer solution in the rear end of latter half
The connecting hole 29 of storage container connection.
The washing device at work, is put into agilent company 20 μm of SPE sieve plates produced in circular hole 22 first
(small SPE sieve plate) 25 is put into Ai Jieer company 3mm SPE sieve plate produced (big SPE sieve plate) 27, big in circular hole 28
The 3kD dialysis membrane 26 of MYM Bioisystech Co., Ltd production is placed in 27 front end of SPE sieve plate, by the interface 24 of first half with after
After half part splicing, stitching portion is tightly wrapped with sealed membrane, elution pipe 23 should elute pipe 23 through the invention in upper and lower ends, upper end
Elution pipe jack 8 on electrophoresis tank is inserted into elution solution tank 2, the casing connection of the elution pipe 23 and wriggling pump side of lower end,
Eluent enters from upper end elution pipe 23, exports washing device by lower end elution pipe 23 after too small SPE sieve plate 25, and wriggling
ICP-MS is introduced under the flow control of pump.
Illustrate the beneficial effect of technical solution of the present invention below by several specific detection embodiments.
Embodiment 1
Protein standard substance is separated offline using horizontal column gel electrophoresis apparatus of the invention, and with commercialization
Reference result compares.
The separation that filler in column gel tube is the concentration glue that 50 μ l concentration are 4% and 400 μ l concentration are 10%
Glue, added protein standard substance are Bio-Rad company Precision Plus Protein Dual Color egg produced
White standard items.Buffer solution used in electrophoresis tank is Tris- glycine-SDS buffer, and elution solution is 50mM's
NH4NO3Solution, and separated in the case where being segmented voltage.Voltage first segment used is 30 minutes 60V and second segment 5 hours
200V, separating resulting is as shown in Figure 4 A and 4 B shown in FIG., and Fig. 4 A produces Bio-Rad company by column gel electrophoresis apparatus of the present invention
The result that is separated of Precision Plus Protein Dual Color protein standard substance；Fig. 4 B is Bio-Rad company
Provided SDS-PAGE separating effect figure.It can be seen from the figure that using column gel electrophoresis apparatus of the present invention to albumen mark
The result that the band that quasi- product carry out separation formation is separated with commercialization SDS-PAGE is coincide very much, it was demonstrated that the present invention has good
Protein Separation ability.
Embodiment 2
Using horizontal column gel electrophoresis apparatus of the invention to two kinds with I127The standard protein of label is separated online
And it is detected with ICP-MS combination.
The polyacrylamide gel filled in gel tube used is the concentration glue and 180 μ l concentration of 40 μ l concentration 4%
10% separation gel, I used127Two kinds of standard proteins of label are purchased from Sigma Co., USA, and sample applied sample amount is 20 μ L, egg
White concentration is that 0.01 μ g/ μ L electrophoretic voltage program is 100V 10min, 200V 20min, 600V 2h.The running buffer salt used
Solution are as follows: Tris- glycine-SDS solution.Protein eluate is the NH of 50mM4NO3Solution.
The revolving speed of external peristaltic pump is that 140 μ L/min, used ICP-MS are agilent company 8800 type produced
ICP-MS.Incident power is 1500W, and collision gas flow velocity is 15.0L/min, flow rate of carrier gas 0.80L/min, and secondary air speed is
0.80L/min, fog chamber's temperature are 2 DEG C, and mass number (m/z) is 127I, and the time of integration is 0.1 second.
For testing result as shown in figure 5, peak 1 is RA albumen in figure, peak 2 is CA albumen.It can be seen from the figure that using this hair
Bright column gel electrophoresis apparatus can be fast and accurately to two kinds with I127The standard protein of label is separated and is detected online,
And peak type is preferable, and dead volume is smaller.
Used GE-ICP-MS on-line coupled system as shown in Figure 1, the GE-ICP-MS on-line coupled system to electrophoresis
Instrument gel tube and washing device are transformed, and the glass-gel tube that original internal diameter is 6.5mm is changed to the stone that internal diameter is 2.5mm
English pipe 5 to reduce sample applied sample amount, while improving separating degree and resolution ratio；Using the nylon sleeve of 3D printing as elution
Liquid drainage device 8 changes 9 size of bottom filter core small for 8.2mm, reduction solution dead volume, to improve separating degree.Protein sample
It is controlled by PEEK liquid phase pipeline threeway 11 through peristaltic pump 12 after separation elution and introduces ICP-MS respectively with 140 μ L/min flow velocitys
13 and fraction collector 14.Sample enters ICP-MS through fog chamber and detects to metallic element and nonmetalloid.Fraction collection
Device collects sample and carries out subsequent Identification of Fusion Protein and other detections.
Embodiment 3
As shown in Fig. 6 A, 6B, using horizontal column gel electrophoresis apparatus of the invention to three kinds with I127The standard egg of label
It is white to be separated and detected with ICP-MS combination online respectively, and the commercialization column by optimization is used under similarity condition
Shape gel electrophoresis apparatus detects same standard albumen, wherein since metallic element may be contained in albumen, it is same
When have detected I127And Zn65Signal, and result is compared.
The polyacrylamide gel filled in gel tube used be 50 μ l concentration 4% concentration glue and 200 μ l it is dense
The separation gel of degree 10%, I used127Three kinds of standard proteins of label are purchased from Sigma Co., USA, and sample applied sample amount is 20 μ L,
Protein concentration is that 0.01 μ g/ μ L electrophoretic voltage program is 100V 10min, 200V 20min, 600V 2h.The running buffer used
Salting liquid are as follows: Tris- glycine-SDS solution.Protein eluate is the NH of 50mM4NO3Solution.
The revolving speed of external peristaltic pump is that 140 μ L/min, used ICP-MS are agilent company 8800 type produced
ICP-MS.Incident power is 1500W, and collision gas flow velocity is 15.0L/min, flow rate of carrier gas 0.80L/min, and secondary air speed is
0.80L/min, fog chamber's temperature are 2 DEG C, and mass number (m/z) is 127I, and the time of integration is 0.1 second.
For testing result as shown in Fig. 6 A, Fig. 6 B, Fig. 6 A is separation and testing result of the present apparatus to three kinds of albumen, and Fig. 6 B is
Separation and testing result of the commercialization electrophoretic apparatus to three kinds of albumen after optimization, peak 1 is RA albumen in figure, and peak 2 is CA albumen,
Peak 3 is BSA albumen.It can be seen from the figure that using column gel electrophoresis apparatus of the present invention can fast and accurately to three kinds with
I127The standard protein of label is separated and is detected online, and peak type and response are all better than the commercialization electrophoresis after optimization
Device, this shows that electrophoretic apparatus and its component of the invention have better Protein Separation efficiency and sensitivity for analysis.
Show the horizontal column gel electrophoresis apparatus for Protein Separation of the invention by gel by test of many times verifying
Bore changes small, can not only reduce sample applied sample amount, repeatedly the experimental results showed that smaller internal diameter can be improved albumen
Separating degree and electrophoretic resolution, and separation condition range of choice is wide, but too small internal diameter is unfavorable for the filling of gel；It utilizes
The horizontal strip electrophoresis slot structure of the invention of 3D printing is simple, cheap, more easily operated than existing commercialization electrophoresis tank；Utilize 3D
The washing device of the invention of printing is cheap, and manufacturing process is simple, the experimental results showed that the washing device dead volume is significantly
It reduces, therefore significantly improves detection efficiency and the sensitivity of albumen, and may be implemented disposable or be recycled.It is logical
It crosses and is used cooperatively column gel electrophoresis apparatus of the present invention and its column gel tube, electrophoresis tank and the washing device that are included, and
With the Instrument crosslinkings such as ICP-MS, fast and accurately separation and the online separation and detection offline of protein can be realized.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical scheme and beneficial effects
Describe in detail bright, it should be understood that the above is only a specific embodiment of the present invention, is not intended to restrict the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in protection of the invention
Within the scope of.
Claims (9)
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| CN109085231A (en) * | 2018-08-31 | 2018-12-25 | 广州市众为生物技术有限公司 | A kind of new type gel electrophoresis apparatus |
| CN109374717B (en) * | 2019-01-14 | 2019-04-16 | 上海宝藤生物医药科技股份有限公司 | Gel electrophoresis system, preparation method and method for separating and detecting low-density lipoprotein by using gel electrophoresis system |
| CN113292632B (en) * | 2021-06-30 | 2023-07-11 | 钱智勇 | Method for extracting and purifying exogenous protein of transgenic plant |
| CN117074590B (en) * | 2023-08-25 | 2025-10-31 | 中国科学院生态环境研究中心 | DNA modification detection method by coupling gel electrophoresis with liquid chromatography-mass spectrometry |
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