CN106749504A - For the horizontal column gel electrophoresis apparatus of Protein Separation - Google Patents
For the horizontal column gel electrophoresis apparatus of Protein Separation Download PDFInfo
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- CN106749504A CN106749504A CN201611101473.6A CN201611101473A CN106749504A CN 106749504 A CN106749504 A CN 106749504A CN 201611101473 A CN201611101473 A CN 201611101473A CN 106749504 A CN106749504 A CN 106749504A
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- column gel
- wash
- tank
- washing device
- gel electrophoresis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/24—Extraction; Separation; Purification by electrochemical means
- C07K1/26—Electrophoresis
Abstract
A kind of horizontal column gel electrophoresis apparatus for Protein Separation, the horizontal column gel electrophoresis apparatus use horizontal structure, including：Column gel tube, filled with the gel for for protein sample separate in it；The length of the column gel tube is 8 12cm, preferably 8cm；And electrophoresis tank, cushioning liquid groove of the electrophoresis tank including side of the positive electrode, the cushioning liquid groove of negative side, bosh and the wash-out solution tank positioned at side of the positive electrode positioned at centre；The position for laying the column gel tube is provided with the electrophoresis tank.Column gel electrophoresis apparatus of the invention can be used for the offline separation of different molecular weight albumen and ON-LINE SEPARATION and detection according to the difference of the polyacrylamide filled and with reference to instruments such as ICP MS, and it is short with analysis time, low cost, manufacturing process is simple, dead volume is small, and separation condition range of choice is wide, and separating degree is high, the features such as being reequiped according to actual needs, and with the possibility continued to optimize.
Description
Technical field
The invention belongs to biochemistry detection and analysis technical field, relate more specifically to a kind of level for Protein Separation
Column gel electrophoresis apparatus.
Background technology
Protein as organism basic component, be the executor of the various critical functions of organism, to protein
Composition, the research of 26S Proteasome Structure and Function have 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 include 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 obvious zone of many bars 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) with albumino reaction and make protein inactivation, protein can be changed into that molecular weight is different and the similar material of structure, therefore nothing
By albumen property how, can according to molecular weight difference and be separated, the small albumen of mass-to-charge ratio is soon, far mobile, matter
Lotus is more mobile slowly than big albumen.SDS-PAGE is simple to operate flexibly, high resolution, and favorable reproducibility is the most widely used
Protein stripping technique.
Device form according to holder is different, and gel electrophoresis can be divided into flat electrophoresis, vertical board-like electrophoresis and post again
Shape electrophoresis.Wherein column gel is more beneficial for the collection after the concentration and separation of sample and the combination with subsequent detection system.
Column gel electrophoresis can according to demand select the glass tube of different inner diameters and length, and filling agarose can be used for point in glass tube
Freestone acid, filled polypropylene acrylamide gel can be used to separate protein.By the protein after polyacrylamide electrophoresis separation by wash-out
Device is eluted, and is analyzed and is detected with the combination of icp mses (ICP-MS) technology.Column is coagulated
Gel electrophoresis have been successfully applied to ON-LINE SEPARATION DNA, RNA and small-molecule substance with the combination of ICP-MS technologies.
But existing commercialization column gel electrophoresis apparatus although to can be used for polypeptide, RNA, DNA and albumen etc. more
The separation and detection of material are planted, universality is good, but also causes the internal diameter of its gel column larger, and washing device dead volume is larger, because
This whole polyacrylamide electrophoresis device is all relatively low to the separative efficiency and sensitivity for analysis of albumen, and cost is high, operates more complicated.
It is the key factor for influenceing protein analysis and detection how polyacrylamide electrophoresis device to be optimized and improved.
3D printing technique is a kind of technology completely contradicted with traditional material processing method, and the technology 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 manufacture 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 is that it can almost produce the article of any shape, and eliminating traditional handicraft needs multiple tracks to process
The tedious procedure of program, the manufacturing cycle is short, low cost.In recent years, 3D printing technique was used as an advanced manufacture for frontier nature
Technology fast development, progressively played an important role in the field such as industry manufacture, biomedical, building manufacture, culture and arts.
But not yet there is the precedent that 3D printing technique is applied to column gel electrophoresis apparatus at present.
The content of the invention
In view of this, it is a primary object of the present invention to provide a kind of horizontal column gel electrophoresis dress for Protein Separation
Put, to solve at least one of above-mentioned technical problem.
To achieve the above object, as one aspect of the present invention, the invention provides a kind of water for Protein Separation
Flat column gel electrophoresis apparatus, the horizontal column gel electrophoresis apparatus use horizontal structure, it is characterised in that including：
Column gel tube, filled with the gel for for protein sample separate in it；The length of the column gel tube is 8-
12cm, preferably 8cm；And
Electrophoresis tank, the cushioning liquid groove of the electrophoresis tank including side of the positive electrode, the cushioning liquid groove of negative side, positioned at middle
Bosh and the wash-out solution tank positioned at side of the positive electrode；The position for laying the column gel tube is provided with the electrophoresis tank.
Wherein, the external diameter of the column gel tube is 4-6mm, preferably 4mm；The internal diameter of the column gel tube is 2-
2.2mm, preferably 2.2mm.
Wherein, the electrophoresis tank is manufactured by 3D printing technique；
Preferably, the electrophoresis tank is made of macromolecule polymer material, preferably it is made up of PLA plastics.
Wherein, the depth of the electrophoresis tank is 3.5-5cm, and width is 8-12cm, and total length is 18-22cm；
Preferably, the length of bosh is 5.5-9cm, preferably 5.5cm in the electrophoresis tank；The buffering of side of the positive electrode
The length of solution tank and wash-out solution tank is 5-6cm, preferably 6cm；The length of the cushioning liquid groove of negative side is 5-6cm, excellent
Elect 5cm as.
Wherein, the cushioning liquid groove of the side of the positive electrode wall adjacent with wash-out solution tank is provided with wash-out pipe jack, in institute
The outside for stating the cushioning liquid groove of side of the positive electrode and cushioning liquid groove the same side of negative side is respectively arranged with the electrode of positive and negative electrode
Seat, and equipped with electrode；
Preferably, the top outside the bosh side is provided with movable clamp, for fixing column gel tube
With closing bosh；The opposite side of described movable clamp one end and bosh is respectively arranged with two and tightens screw hole, makes
Used time is used to insert screw tightening activity clamp.
Wherein, the horizontal column gel electrophoresis apparatus also include washing device, and the washing device includes combinable
Former and later two parts；
Preferably, the front end of the first half of the washing device is provided with 6-8mm long, a diameter of 5.5-7.5mm
The jack of column gel tube；The centre of first half is 1.3mm long, the circular hole of a diameter of 4mm, for placing a SPE sieve plates,
Connected by a wash-out pipe respectively in the both sides of circular hole；The rear end of first half is provided with 4.1mm long, diameter 11.2mm
Interface connects with latter half；The front end of the latter half of the washing device is provided with 1.6mm long, the circle of a diameter of 9.2mm
Hole, for placing the 2nd SPE sieve plates, dialysis membrane is placed with the 2nd SPE sieve plates front end；Set in the rear end of latter half
There is the connecting hole communicated with cushioning liquid storage container.
Wherein, the main part of the washing device uses macromolecule polymer material, preferably synthetic resin to be made；
Preferably, the washing device main part is manufactured by 3D printing technique.
Wherein, column gel tube is inserted into the first half of the washing device when in use and contacts described first
SPE sieve plates；
Preferably, be bolted together for former and later two parts when in use by the washing device, and tightly wrapped with sealed membrane
Interface.
Wherein, the washing device is abundant to a SPE sieve plates, the 2nd SPE sieve plates and dialysis membrane using preceding needs
Wetting, and air is excluded with order to be effective.
Wherein, wash-out pipe should be in upper and lower ends when the washing device is used, and upper end wash-out pipe is by washing on electrophoresis tank
De- pipe jack is inserted into wash-out solution tank, and the wash-out pipe of lower end is eluted with the sleeve pipe connection of wriggling pump side, eluent from upper end
Pipe enters, and by deriving washing device by lower end wash-out pipe after a SPE sieve plates, and electricity is introduced under the flow control of peristaltic pump
Sense couple plasma mass spectrometer.
Understand that horizontal column gel electrophoresis apparatus of the invention have the advantages that based on such scheme：(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 analytical instrument such as device combination ICP-MS can realize protein sample fast and accurately ON-LINE SEPARATION and detection, improve dividing for albumen
From efficiency and the sensitivity of analysis；(3) column gel tube of the invention coordinates according to the difference of the polyacrylamide gel of filling
Electrophoresis tank uses the quick separating for being capable of achieving different proteins；(4) horizontal column gel electrophoresis apparatus main body of the invention is used
3D printing is manufactured, and material therefor is about 300 grams, about 500 yuans or so of cost, whole manufacturing process less than 18 hours,
Light weight, with low cost, manufacturing process is fast and simple；(5) washing device dead volume of the invention is small, cheap, is capable of achieving
Disposable or reuse；(6) column gel electrophoresis apparatus simple structure of the invention, it is easy to operate；(7) gel of the invention
Electrophoretic apparatus have Protein Separation efficiency higher, and with low cost, manufacturing process it is simple, dead volume is small, quick and precisely
The features such as, and with the possibility for continuing to optimize.
Brief description of the drawings
Fig. 1 is the structural representation of column gel tube of the invention；
Fig. 2 is the structural representation of electrophoresis tank of the invention；
Fig. 3 A and Fig. 3 B are respectively the profile of washing device left-half of the invention and the STRUCTURE DECOMPOSITION of washing device
Schematic diagram；
Fig. 4 A and Fig. 4 B are respectively to use column gel electrophoresis Protein Separation device (column gel tube and electrophoresis of the invention
Groove) the Precision Plus Protein Dual Color protein standard substances that are produced to Bio-Rad companies are separated
The separating effect figure of the SDS-PAGE that result is provided with Bio-Rad companies；
Fig. 5 is to use gel electrophoresis Protein Separation device combination ICP-MS of the invention to two kinds with I127The standard of mark
Albumen carries out the electrophoresis result figure of ON-LINE SEPARATION and detection, and wherein peak 1 is RA albumen, and peak 2 is CA albumen.
Fig. 6 A are to use gel electrophoresis Protein Separation device combination ICP-MS of the invention to three kinds with I127The standard of mark
Albumen carries out the electrophoresis result figure of ON-LINE SEPARATION and detection, and Fig. 6 B are in same batten using the commercialization electrophoretic apparatus by optimization
The result figure detected to three kinds of same standard proteins under part, wherein peak 1 are RA albumen, and peak 2 is CA albumen, and peak 3 is BSA
Albumen.
In upper figure, reference implication is as follows：
1.-- cushioning liquid grooves；2.-- elutes solution tank；3.-- boshes；4.-- negative electrodes；5.-- positive electrodes；6.--
Wire electrode groove；7.-- gel tube placed holes；8.-- elutes pipe jack；9.-- cooling water inlets；10.-- coolant outlets；11.--
Movable clamp；12.-- tightens screw hole；
21.-- gel tube jacks；The small SPE sieve plates putting holes of 22.--；23.-- wash-out pipes；24.-- interfaces；25.--20μm
SPE sieve plates (small SPE sieve plates)；26.--3kD dialysis membranes；27.--3mm SPE sieve plates (big SPE sieve plates)；The big SPE sieve plates of 28.--
Putting hole；29.-- cushioning liquid intercommunicating pores.
Specific embodiment
To make the object, technical solutions and advantages of the present invention become more apparent, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in further detail.
The present invention relates to a kind of horizontal column gel electrophoresis apparatus for Protein Separation, the device is mainly solidifying comprising column
Sebific duct, three parts of electrophoresis tank and washing device.Wherein, electrophoresis groove body and washing device main body are manufactured using 3D printing, material
Matter is macromolecular material, for example PLA plastics and synthetic resin, and it has the following advantages that：According to the situation of actual sample, using this
Horizontal column gel electrophoresis apparatus and its column gel tube and electrophoresis tank that are included, can be used for the offline separation of albumen；Use
The horizontal column gel electrophoresis apparatus and its column gel tube, electrophoresis tank and washing device for being included, with reference to ICP-MS etc.
Analytical instrument, can be used for the ON-LINE SEPARATION of albumen and detection.
When specifically used, when offline inspection is needed, different polyacrylamide gels are filled in column gel tube, to
Protein sample to be separated is added in column gel tube, and is placed in electrophoresis tank, then in the different slots of gel electrophoresis groove
Cushioning liquid and cooling circulating water, fastening activity clamp are separately added into, and the electrode of electrophoresis tank is switched on power, afterwards albumen sample
Product can offline be separated in the presence of electric current in gel tube, and whole process is easy to operate, and separative efficiency is high.
When on-line checking is needed, different polyacrylamide gels are filled in column gel tube, by column gel tube
It is placed in electrophoresis tank, and is connected with washing device, the wherein side wash-out pipe of washing device passes through peristaltic pump and inductive
Plasma mass spectrograph injection port is connected, and to adding protein sample to be separated in column gel tube, and is placed in electrophoresis tank,
Then to be separately added into the different slots of gel electrophoresis groove cushioning liquid, wash-out solution and cooling circulating water, fastening activity clamp,
And the electrode of electrophoresis tank switches on power, protein sample can be separated in the presence of electric current in gel tube afterwards, and with
Eluent is analyzed and detects in the presence of peristaltic pump into ICP-MS.So can simultaneously realize that protein sample is quickly accurate
True ON-LINE SEPARATION and detection, and whole device is easy to operate, and dead volume is small, separates and detection efficiency is high.
Specifically, horizontal column gel electrophoresis apparatus of the invention include column gel tube, electrophoresis tank and selectable wash
De- device.
Wherein, the horizontal column gel electrophoresis apparatus use horizontal structure, i.e. column gel tube horizontal positioned.Column gel
Different types of polyacrylamide gel can be filled in pipe, is needed to select correspondence gel according to detection.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
Need to separate molecular weight protein wide, then can fill gradient gel.By validation trial and accounting, the length of column gel tube
Can be according to gel filled how much settings, for example, 8-12cm, external diameter is then preferably 4-6mm, more preferably 4mm, internal diameter
Preferably 2-2.2mm, more preferably 2.2mm, so as to significantly improve the separative efficiency for albumen.
Wherein, the cushioning liquid groove of electrophoresis tank including positive and negative electrode side, positioned at middle bosh and positioned at side of the positive electrode
Wash-out solution tank.The cushioning liquid groove of the side of the positive electrode wall adjacent with wash-out solution tank is provided with wash-out pipe jack, slow at two
Rush the outside of solution tank side has the electrode tip holder of positive and negative electrode respectively, and equipped with electrode, the conducting Pt wire being connected with electrode passes through
Aperture on groove extends to two cushioning liquid trench bottoms along wire electrode draw-in groove, in two cushioning liquid grooves and bosh phase
There is gel tube placed hole on the wall for connecing respectively, bosh is respectively provided on two sides with cooling water inlet and coolant outlet, cooling
Tank side outer tip end has movable clamp, for immobilized gel pipe and closing bosh, movable clamp one end and cooling water
Groove opposite side has two to tighten screw hole respectively, is used to insert screw tightening activity clamp when using.
Preferably, electrophoresis tank uses macromolecule polymer material, such as PLA plastics to be made.It is further preferred that electrophoresis
Groove 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 bosh is, for example, 5.5-9cm in electrophoresis tank, preferably 5.5cm；Side of the positive electrode cushioning liquid groove
Length with wash-out solution tank is, for example, 5-6cm, preferably 6cm；The length of negative side cushioning liquid groove is, for example, 5-6cm, excellent
Elect 5cm as.
Preferably, the diameter of the gel tube placed hole on cushioning liquid cell wall in electrophoresis tank is, for example, 4mm.
Wherein, with life between the movable clamp of the bosh side outer tip end in electrophoresis tank and corresponding electrophoresis cell wall
Material strip is wound to avoid leak, and two when using with screw insertion activity clamp one end and bosh opposite side respectively tighten
Tightening movable clamp in screw hole.
Washing device includes combinable former and later two parts, and the front end of first half is provided with 6-8mm long, a diameter of
The jack of the column gel tube of 5.5-7.5mm, added with rubber hose to ensure air-tightness, the centre of first half is length for centre
The circular hole of 1.3mm, a diameter of 4mm, for placing 20 μm of SPE sieve plates (small SPE sieve plates) that Agilent company is produced, in circle
The both sides in hole are connected by a wash-out pipe respectively, and the rear end of first half is provided with the interface of 4.1mm long, diameter 11.2mm
Connect with latter half.The front end of latter half is provided with 1.6mm long, the circular hole of a diameter of 9.2mm, public for placing Ai Jieer
The produced 3mm SPE sieve plates (big SPE sieve plates) of department, the production of MYM Bioisystech Co., Ltd is placed with big SPE sieve plates front end
3kD dialysis membranes, the connecting hole communicated with cushioning liquid storage container is provided with the rear end of latter half.
Preferably, the main part of washing device is for example with macromolecule polymer material, such as synthetic resin is made.Enter
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 plates.In another preferred embodiment, washing device is when in use by former and later two parts
It is bolted together, and interface is tightly wrapped with sealed membrane.
Preferably, washing device is using preceding needing fully to soak large and small SPE sieve plates and dialysis membrane, and exclude air
With in order to be effective.
Preferably, washing device elutes pipes respectively positioned at upper and lower ends for two when in use, the wash-out pipe of upper end passes through
Wash-out pipe jack on electrophoresis tank is inserted into wash-out solution tank, the wash-out pipe and the sleeve pipe connection of wriggling pump side of lower end, and
Icp mses are introduced under the flow control of peristaltic pump.
When the horizontal column gel electrophoresis apparatus are used for the offline separation of albumen, need to simultaneously using column gel tube and
Two parts of electrophoresis tank；When the horizontal column gel electrophoresis apparatus are used for the ON-LINE SEPARATION of albumen, need to be simultaneously solidifying using column
Sebific duct, three parts of electrophoresis tank and washing device.
The invention also discloses a kind of gel electrophoresis albumen point using horizontal column gel electrophoresis apparatus as described above
From detecting system.
As a preferred embodiment, the invention discloses a kind of horizontal column gel electrophoresis dress for Protein Separation
Put, the device includes column gel tube, three parts of electrophoresis tank and washing device, 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 CubePro 3D desktops level printer printing that Systems companies are produced, printed material is PLA plastics, consumptive material about 300g, consumption
When about 18 hours.Washing device main body is printed using the Form2 desktops level 3D printer that Formlabs companies are produced, printing
Material is synthetic resin, and consumptive material about 5g takes about 1 hour.
Another preferred embodiment of the present invention is described further below in conjunction with the accompanying drawings.
As shown in figure 1, this is used for column gel length of tube of Protein Separation for 8cm, external diameter are 4mm, internal diameter is 2.2mm.
The column gel tube operationally, it is necessary to inside filling different volumes and concentration polyacrylamide gel, it
It is placed into afterwards on the gel tube placed hole on electrophoresis tank of the invention, makes with reference to electrophoresis tank of the invention and washing device etc.
With after adding protein sample and being powered, protein sample can be moved and divided in the presence of electric current from negative pole to positive pole
From.
As shown in Fig. 2 the column gel electrophoresis groove for being used for Protein Separation uses PLA plastic manufacturings, full weight to be about 300
Gram, the electrophoresis groove depth is 4cm, and width is 10cm, and total length is 20cm.The electrophoresis tank includes：Two of positive and negative electrode side delay
Rush solution tank 1, wash-out solution tank 2, bosh 3, negative electrode 4, positive electrode 5, wire electrode groove 6, gel tube placed hole 7, wash-out
Pipe jack 8, cooling water inlet 9, coolant outlet 10, movable clamp 11, tighten screw hole 12.Electrophoresis tank includes four grooves, point
It is not the wash-out solution tank 2 of two cushioning liquid grooves 1, middle bosh 3 and side of the positive electrode of positive and negative electrode side, wherein cold
But the length of tank 3 is 5.5cm, and the cushioning liquid groove 1 of side of the positive electrode and the length of wash-out solution tank 2 are 6cm, the buffering of negative side
The length of solution tank 1 is 5cm.Have positive and negative electrode 4,5 respectively in the outside of two cushioning liquid groove sides, positive and negative electrode side it is slow
Rush the side adjacent with bosh 3 of solution tank 1 has gel tube placed hole 7 respectively, its a diameter of 4mm, with negative electrode 4 and positive electricity
The conducting Pt wire of the connection of pole 5 is laid on the bottom of the cushioning liquid groove 1 of both positive and negative polarity side along wire electrode groove 6 respectively, side of the positive electrode it is slow
Rush the wall adjacent with wash-out solution tank 2 of solution tank 1 and be provided with wash-out pipe jack 8.The both sides of bosh 3 are provided with cooling water inlet
9 and coolant outlet 10, there is movable clamp 11 bosh side, for immobilized gel pipe and closing bosh 3, active card
The one end of plate 11 and the opposite side of bosh 3 have two to tighten screw hole 12 respectively, for tightening movable clamp 11.
The electrophoresis tank operationally, is separately added into the slow of 150ml or so in two cushioning liquid grooves 1 of positive and negative electrode side
Solution is rushed, by 8cm long, external diameter is placed in gel tube placed hole 7 for the column gel tube of 4mm, then detains movable clamp 11
On, fixed by tightening screw hole 12 with two screws, wherein being wrapped with raw material band to avoid Lou between movable clamp and electrophoresis tank
Liquid, be connected on power supply for negative electrode 4 and positive electrode 5 respectively by electric lead, and appropriate wash-out solution is added in solution tank 2 is eluted,
The washing device one end for using cooperatively.
As shown in Fig. 3 A, 3B, the washing device for being used for Protein Separation includes combinable former and later two parts, first half
Point front end be provided with a length of 8mm, the jack 21 of the column gel tube of a diameter of 5.6mm, it is middle added with rubber hose ensureing
Air-tightness, the centre of first half is the circular hole 22 of 1.3mm long, diameter 4mm, for placing 20 μm that Agilent company is produced
SPE sieve plates (small SPE sieve plates) 25, have a wash-out pipe to connect respectively in the both sides of circular hole, are provided with the rear end of first half
A length of 4.1mm, the interface 24 of a diameter of 11.2mm connect with latter half.The front end of latter half is provided with 1.6mm long, diameter
It is the circular hole 28 of 9.2mm, for placing the 3mm SPE sieve plates (big SPE sieve plates) 27 that Ai Jieer companies are produced, before big sieve plate
End is placed with the 3kD dialysis membranes 26 of MYM Bioisystech Co., Ltd production, is provided with and cushioning liquid in the rear end of latter half
The connecting hole 29 of storage container connection.
The washing device operationally, first in circular hole 22 is put into 20 μm of SPE sieve plates that Agilent company is produced
(small SPE sieve plates) 25, is put into the 3mm SPE sieve plates (big SPE sieve plates) 27 that Ai Jieer companies are produced, big in circular hole 28
The 3kD dialysis membranes 26 of MYM Bioisystech Co., Ltd production are placed in the front end of SPE sieve plates 27, by the interface 24 of first half with it is rear
After half part splicing, stitching portion is tightly wrapped with sealed membrane, wash-out pipe 23 should be in upper and lower ends, and upper end wash-out pipe 23 is by of the invention
Wash-out pipe jack 8 on electrophoresis tank is inserted into wash-out solution tank 2, the wash-out pipe 23 and the sleeve pipe connection of wriggling pump side of lower end,
Eluent enters from upper end wash-out pipe 23, derives washing device by lower end wash-out pipe 23 after too small SPE sieve plates 25, and wriggling
ICP-MS is introduced under the flow control of pump.
The beneficial effect of technical solution of the present invention is illustrated 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 is contrasted.
Filler in column gel tube is the concentration glue that 50 μ l concentration are 4% and the separation that 400 μ l concentration are 10%
Glue, the Precision Plus Protein Dual Color eggs that the protein standard substance for being added is produced by Bio-Rad companies
White standard items.Cushioning liquid used in electrophoresis tank is Tris- glycine-SDS buffer solutions, and wash-out solution is 50mM's
NH4NO3Solution, and separated in Segmented electrical pressure.Voltage first paragraph used is 30 minutes 60V and second segment 5 hours
200V, as shown in Figure 4 A and 4 B shown in FIG., Fig. 4 A are produced separating resulting by column gel electrophoresis apparatus of the present invention to Bio-Rad companies
The separate result of Precision Plus Protein Dual Color protein standard substances；Fig. 4 B are Bio-Rad companies
The SDS-PAGE separating effect figures for being provided.It can be seen that using column gel electrophoresis apparatus of the present invention to albumen mark
The band that quasi- product separate formation is very identical with the result that commercialization SDS-PAGE is separate, 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 mark carries out ON-LINE SEPARATION
And detected with ICP-MS combinations.
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 mark 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 voltages program is 100V 10min, 200V 20min, 600V 2h.The running buffer salt for using
Solution is：Tris- glycine-SDS solution.Protein eluate is the NH of 50mM4NO3Solution.
The rotating speed of external peristaltic pump is 140 μ L/min, 8800 types that the ICP-MS for being used is produced by Agilent company
ICP-MS.Incident power is 1500W, and collision gas flow velocity is 15.0L/min, and flow rate of carrier gas is 0.80L/min, and secondary air speed is
0.80L/min, fog chamber's temperature is 2 DEG C, and mass number (m/z) is 127I, and the time of integration is 0.1 second.
Testing result as shown in figure 5, in figure peak 1 be RA albumen, peak 2 be CA albumen.It can be seen that using this hair
Bright column gel electrophoresis apparatus can be fast and accurately to two kinds with I127The standard protein of mark carries out ON-LINE SEPARATION and detection,
And preferably, dead volume is smaller for peak type.
The GE-ICP-MS on-line coupled systems for being used are as shown in figure 1, the GE-ICP-MS on-line coupled systems are to electrophoresis
Instrument gel tube and washing device are transformed, by original internal diameter for the glass-gel tube of 6.5mm is changed to the stone that internal diameter is 2.5mm
English pipe 5, so that sample applied sample amount is reduced, while improving separating degree and resolution ratio；By the use of the nylon sleeve of 3D printing as wash-out
It is 8.2mm that liquid drainage system 8 changes small the size of bottom filter core 9, reduces solution dead volume, so as to improve separating degree.Protein sample
Control to introduce ICP-MS respectively with 140 μ L/min flow velocitys through peristaltic pump 12 by PEEK liquid phase pipelines threeway 11 after separating wash-out
13 and fraction collector 14.Sample is detected into ICP-MS through fog chamber to metallic element and nonmetalloid.Fraction collection
Device collects sample and carries out follow-up 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 mark
It is white to carry out ON-LINE SEPARATION respectively and detected with ICP-MS combinations, and the commercialization post by optimization is used under similarity condition
Shape gel electrophoresis apparatus detect to same standard albumen, wherein due to metallic element may be contained in albumen, therefore together
When have detected I127And Zn65Signal, and result is compared.
The concentration glue and 200 μ l that the polyacrylamide gel filled in gel tube used is 50 μ l concentration 4% are dense
The separation gel of degree 10%, I used127Three kinds of standard proteins of mark 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 voltages program is 100V 10min, 200V 20min, 600V 2h.The running buffer for using
Salting liquid is：Tris- glycine-SDS solution.Protein eluate is the NH of 50mM4NO3Solution.
The rotating speed of external peristaltic pump is 140 μ L/min, 8800 types that the ICP-MS for being used is produced by Agilent company
ICP-MS.Incident power is 1500W, and collision gas flow velocity is 15.0L/min, and flow rate of carrier gas is 0.80L/min, and secondary air speed is
0.80L/min, fog chamber's temperature is 2 DEG C, and mass number (m/z) is 127I, and the time of integration is 0.1 second.
As shown in Fig. 6 A, Fig. 6 B, Fig. 6 A are separation and testing result of the present apparatus to three kinds of albumen to testing result, and Fig. 6 B are
The separation and testing result of commercialization electrophoretic apparatus after optimization to three kinds of albumen, peak 1 is RA albumen in figure, and peak 2 is CA albumen,
Peak 3 is BSA albumen.It can be seen that using column gel electrophoresis apparatus of the present invention can fast and accurately to three kinds with
I127The standard protein of mark carries out ON-LINE SEPARATION and detection, and peak type and response are all better than the commercialization electrophoresis after optimization
Device, this shows that electrophoretic apparatus of the invention and its part have more preferable Protein Separation efficiency and sensitivity for analysis.
Show that the horizontal column gel electrophoresis apparatus for Protein Separation of the invention are by gel by test of many times checking
Bore changes small, can not only reduce sample applied sample amount, repeatedly test result indicate that smaller internal diameter can improve 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；Utilize
The Horizontal electrophoresis tank simple structure of the invention of 3D printing, it is cheap, it is more easily operated than existing commercialization electrophoresis tank；Using 3D
The washing device of the invention of printing is cheap, and manufacturing process is simple, test result indicate that the washing device dead volume is significantly
Reduce, therefore significantly improve detection efficiency and the sensitivity of albumen, it is possible to realize single use or recycling.It is logical
Cross and use 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 crosslinking such as ICP-MS, can realize that the fast and accurately offline of protein is separated and ON-LINE SEPARATION and detection.
Particular embodiments described above, has been carried out further in detail to the purpose of the present invention, technical scheme and beneficial effect
Describe in detail bright, it should be understood that the foregoing is only specific embodiment of the invention, be not intended to limit the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc. should be included in protection of the invention
Within the scope of.
Claims (10)
1. a kind of horizontal column gel electrophoresis apparatus for Protein Separation, the horizontal column gel electrophoresis apparatus use level
Structure, it is characterised in that including：
Column gel tube, filled with the gel for for protein sample separate in it；The length of the column gel tube is 8-12cm,
Preferably 8cm；And
Electrophoresis tank, the electrophoresis tank includes cushioning liquid groove, the cushioning liquid groove of negative side, the cooling positioned at centre of side of the positive electrode
Tank and the wash-out solution tank positioned at side of the positive electrode；The position for laying the column gel tube is provided with the electrophoresis tank.
2. horizontal column gel electrophoresis apparatus according to claim 1, it is characterised in that the external diameter of the column gel tube
It is 4-6mm, preferably 4mm；The internal diameter of the column gel tube is 2-2.2mm, preferably 2.2mm.
3. horizontal column gel electrophoresis apparatus according to claim 1, it is characterised in that the electrophoresis tank passes through 3D printing
Technology is manufactured；
Preferably, the electrophoresis tank is made of macromolecule polymer material, preferably it is made up of PLA plastics.
4. horizontal column gel electrophoresis apparatus according to claim 1, it is characterised in that the depth of the electrophoresis tank is
3.5-5cm, width is 8-12cm, and total length is 18-22cm；
Preferably, the length of bosh is 5.5-9cm, preferably 5.5cm in the electrophoresis tank；The cushioning liquid of side of the positive electrode
The length of groove and wash-out solution tank is 5-6cm, preferably 6cm；The length of the cushioning liquid groove of negative side is 5-6cm, preferably
5cm。
5. horizontal column gel electrophoresis apparatus according to claim 1, it is characterised in that the cushioning liquid of the side of the positive electrode
The groove wall adjacent with wash-out solution tank is provided with wash-out pipe jack, in the cushioning liquid groove and the buffering of negative side of the side of the positive electrode
The outside of solution tank the same side is respectively arranged with the electrode tip holder of positive and negative electrode, and equipped with electrode；
Preferably, the top outside the bosh side is provided with movable clamp, for fixing column gel tube and envelope
Close bosh；The opposite side of described movable clamp one end and bosh is respectively arranged with two and tightens screw hole, when using
For inserting screw tightening activity clamp.
6. horizontal column gel electrophoresis apparatus according to claim 1, it is characterised in that the horizontal column gel electrophoresis
Device also includes washing device, and the washing device includes combinable former and later two parts；
Preferably, the front end of the first half of the washing device is provided with 6-8mm long, the column of a diameter of 5.5-7.5mm
The jack of gel tube；The centre of first half is 1.3mm long, the circular hole of a diameter of 4mm, for placing a SPE sieve plates, in circle
The both sides in hole are connected by a wash-out pipe respectively；The rear end of first half is provided with the interface of 4.1mm long, diameter 11.2mm
Connect with latter half；The front end of the latter half of the washing device is provided with 1.6mm long, the circular hole of a diameter of 9.2mm, uses
In the 2nd SPE sieve plates are placed, dialysis membrane is placed with the 2nd SPE sieve plates front end；The rear end of latter half be provided with
The connecting hole that cushioning liquid storage container is communicated.
7. horizontal column gel electrophoresis apparatus according to claim 6, it is characterised in that the main part of the washing device
Dividing uses macromolecule polymer material, preferably synthetic resin to be made；
Preferably, the washing device main part is manufactured by 3D printing technique.
8. according to the horizontal column gel electrophoresis apparatus described in claim 6, it is characterised in that when in use insert column gel tube
Enter the first half to the washing device and contact a SPE sieve plates；
Preferably, be bolted together for former and later two parts when in use by the washing device, and interface is tightly wrapped with sealed membrane
Place.
9. according to the horizontal column gel electrophoresis apparatus described in claim 6, it is characterised in that the washing device uses preceding needs
The first SPE sieve plates, the 2nd SPE sieve plates and dialysis membrane are fully soaked, and excludes air with order to be effective.
10. according to the horizontal column gel electrophoresis apparatus described in claim 6, it is characterised in that the washing device is washed when using
De- pipe should be inserted into wash-out solution tank in upper and lower ends, upper end wash-out pipe by the wash-out pipe jack on electrophoresis tank, lower end
The sleeve pipe connection of wash-out pipe and wriggling pump side, eluent enters from upper end wash-out pipe, by being eluted by lower end after a SPE sieve plates
Pipe derives washing device, and introduces icp mses under the flow control of peristaltic pump.
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| CN201611101473.6A CN106749504B (en) | 2016-12-02 | 2016-12-02 | Horizontal column-shaped gel electrophoresis device for protein separation |
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| CN201611101473.6A CN106749504B (en) | 2016-12-02 | 2016-12-02 | Horizontal column-shaped gel electrophoresis device for protein separation |
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| CN108572099A (en) * | 2018-04-23 | 2018-09-25 | 上海交通大学 | A kind of method and apparatus for realizing biological tissue's rapid dyeing |
| CN113292632A (en) * | 2021-06-30 | 2021-08-24 | 钱智勇 | Method for extracting and purifying exogenous protein of transgenic plant |
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| CN106749504B (en) | 2020-05-26 |
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