CN104792851B - Micropreparation-type gel electrophoresis device and use method thereof - Google Patents
Micropreparation-type gel electrophoresis device and use method thereof Download PDFInfo
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Abstract
The invention relates to a micro gel electrophoresis device and a use method thereof. The micro gel electrophoresis device is characterized by comprising a preparation-type gel electrophoresis separation device and a preparation-type gel electrophoresis elution collection device, wherein the preparation-type gel electrophoresis separation device comprises two clamp plates, two gel preparation glass plates, a base and a refrigerating device; the two gel preparation glass plates are arranged in grooves formed in the inner side surfaces of the two clamp plates respectively; the depths of the two grooves are less than the thicknesses of the two gel preparation glass plates; the two clamp plates are placed with the inner side surfaces opposite, locked via screws and then arranged in the base in an inserting manner; the refrigerating device comprises two refrigerating pieces; the two refrigerating pieces are arranged on the outer sides of the two clamp plates respectively, and parallel to vertical planes of the two clamp plates; the preparation-type gel electrophoresis elution collection device comprises two clamp plates, an electrode cover and a collection groove; the two clamp plates are placed with the inner side surfaces opposite, and locked via screws; the electrode cover is arranged above the two clamp plates; and the collection groove is formed below the two clamp plates. The micro gel electrophoresis device can be widely used for proteomics analysis and medicine protein separation and purification.
Description
Technical field
The present invention relates to bioanalysiss and biological medicine research field, especially with regard to a kind of micropreparation type gel electrophoresiss
Device and its using method.
Background technology
The one kind of SDS-PAGE (SDS-PAGE) technology as gel electrophoresis
Pattern, be widely used in biochemistry, appreciation science, molecular biology, proteomics and pharmaceutical grade protein etc. research and
Analysis field.The principle of SDS-PAGE technologies is the difference using protein molecular weight size so as to divided in the gel of electrophoresis
From.Separation of Proteins analysis the most frequently used at present and technology of preparing are gel electrophoresiss and electrophoresis elution.Wherein, preparative gel
Electrophoresis not only can reduce the complexity of sample, enrichment low abundance proteinses before sample analysis, remove high-abundance proteins matter
And separation and concentration post translational modification protein, improve the sensitivity of protein and protein group mass spectral analyses, accuracy and cover
Lid rate, can be also used for isolating and purifying or overall protein (Top-down) mass spectral analyses from top to bottom for some pharmaceutical grade proteins
Front high efficiency separation, thus receive significant attention.
At present, heat dissipation problem is solved with water-cooled using cylinder or Ring-cylindrical device more than preparative gel electrophoresis system,
Semipermeable membrane is placed between gel and positive pole, and certain space is reserved between gel and semipermeable membrane, electrode solution is promoted not using pump
Broken belt is walked the protein isolated from gel and which is collected, so as to reach the purpose of albumen preparation.For example, Chen Jinhai
Invention prepare electrophoresis system and Japanese Scientists Zhenxiong Chi Yan design many sets prepare electrophoresis equipment.It is higher in order to reach
Separating degree, these equipment generally adopt longer separating component, thus its volume is typically huger, while in order to take away gel
The heat of middle generation, generally requires large volume of water cooling system.In addition, in practical operation, operator also need to often
A pump is switched every a period of time, detached protein fraction between gel and positive pole is collected.With the preparation of Zhenxiong Chi Yan
As a example by electrophoresis system, preparing once blood red sample protein needs to collect at least 80 times fractions, and whole acquisition time needs 12 are little
When more than.These shortcomings limit large volume preparative gel electrophoresis system and separate analysis and protein group in general biological sample
Application in.Recently, the commercialization GELFREE 8100 that Britain Expedeon companies release prepares electrophresis apparatuses and also utilizes gel
Electrophoretic techniquess separate and collect the purpose that fraction realizes prepared by electrophoresis, but to there is also separating gel in itself too short for its instrument, point
From the shortcomings of spending low and elution time length and disposable gel high cost.
The content of the invention
For the problems referred to above, it is an object of the invention to provide a kind of device volume it is little, easy to operate, separate and collection efficiency
High micropreparation type gel electrophoresis apparatus and its using method.
For achieving the above object, the present invention takes technical scheme below：A kind of micropreparation type gel electrophoresis apparatus, which is special
Levy and be：It includes preparative gel electrophoresis separating device and preparative gel electrophoresiss eluting collection device；The preparative is coagulated
Gel electrophoresis segregation apparatuss include first clamping plate, second clamping plate, the first glue glass plate, the second glue glass plate, a base and
Refrigerating plant；The first glue glass plate and the second glue glass plate are separately positioned in the first clamping plate and second clamping plate
In the groove that side is arranged, and the depth of groove described in two is respectively less than the first glue glass plate and the second glue glass plate
Thickness；The first clamping plate and second clamping plate medial surface are staggered relatively and by some screw locks, and the first clamping plate and
The boss that second clamping plate bottom is arranged is plugged in the base；The refrigerating plant includes two cooling pieces, cooling piece described in two
The outside of the first clamping plate and second clamping plate is separately positioned on, and is put down with the vertical plane of the first clamping plate and second clamping plate
OK；The preparative gel electrophoresiss eluting collection device includes three-ply board, the 4th clamping plate, an electrode cap and a collecting tank；Institute
The medial surface for stating three-ply board and the 4th clamping plate is staggered relatively and by some screw locks；The electrode cap is arranged on described
The top of three-ply board and the 4th clamping plate, the collecting tank are arranged on the lower section of the three-ply board and the 4th clamping plate.
The first negative electrode liquid bath is provided with the top of the first clamping plate, the first negative electrode liquid bath bottom is provided with
Two first wire columns, are wound with the first platinum electrode silk on the first wire column described in two, and the first platinum electrode silk be arranged on
First negative electricity pole of the first clamping plate upper surface is connected；The medial surface of the first negative electrode liquid bath is provided with first
Opening, first mouth periphery are provided with first " U " type silicon rubber bar；First " U " the type silicon rubber bar be partially submerged in
The medial surface of the first clamping plate, and the enclosed area of first " U " the type silicon rubber bar is slightly larger than the described first opening；Described
One glue glass plate upper end is provided with first opening described with the first negative electrode liquid bath corresponding second and is open.
The base is closed box structure, and which is internally provided with two second wire columns, twines on the second wire column described in two
It is wound with the second platinum electrode silk, and the second platinum electrode silk and the first positive electrical pole phase for being arranged on the base upper surface
Even；The upper surface of the base is additionally provided with the 3rd opening, and the width of the 3rd opening should meet and can put down described first
Clamping plate, second clamping plate, the first glue glass plate and the second glue glass plate.
Some eluting rooms are arranged side by side at the top of the three-ply board, the medial surface of each eluting room is provided with the 4th
Opening；A longitudinal cylindrical channel for leading directly to the three-ply board bottom is provided with below each eluting room；And each institute
State and below eluting room, be additionally provided with a laterally cylindrical collecting tank switch, be provided with the collecting tank switch described vertical with each
To the hole of cylindrical channel matching, for controlling the discharge of each eluting indoor liquid；Also set up in the three-ply board
Have a 3rd platinum electrode silk, the 3rd platinum electrode silk runs through all eluting rooms, and be arranged on the three-ply board surface
The second positive electrical pole be connected.
The second negative electrode liquid bath is provided with the top of 4th clamping plate, the second negative electrode liquid bath bottom is provided with
4th platinum electrode silk, and the 4th platinum electrode silk is connected with the second negative electricity pole for being arranged on the 4th cleat surface；
Some 5th openings are provided with the medial surface of the second negative electrode liquid bath, and each 5th opening is pressed from both sides with the described 3rd respectively
4th opening of each described eluting indoor profile of plate is corresponding；The medial surface of the second negative electrode liquid bath also sets up
There is second " U " type silicon rubber bar, second " U " the type silicon rubber bar is partially submerged in the 4th clamping plate medial surface, and described
The enclosed area of second " U " type silicon rubber bar is slightly larger than the second negative electrode liquid bath；Two points are provided with the electrode cap
The parent part not matched with the three-ply board and the second positive and negative electrode electrode column on the 4th clamping plate；On the collecting tank
The groove matched with each eluting room in the three-ply board is provided with, for the albumen to eluting in each eluting room
Fraction is collected.
A kind of using method of micropreparation type gel electrophoresis apparatus, comprises the following steps：
1), after first, second glue glass plate being inserted respectively into first clamping plate and the corresponding groove of second clamping plate, use spiral shell
Nail locking；2) the corresponding solution of desired concn is prepared, and is poured into the first glue glass of preparative gel electrophoresis separating device
Between glass plate and the second glue glass plate, the gel for forming solid-state in a moment is stood；3) it is open the bottom of to from the 3rd of susceptor surface the
Add electrode buffer in seat, and by first clamping plate, the first glue glass plate, the second glue glass plate and second clamping plate insertion the
In three openings；4) electrode buffer is added in the first negative electrode liquid bath, until electrode buffer passes through the first glue glass
Opening above plate is immersed between the first glue glass plate and the second glue glass plate；To the first glue glass plate and the second system
Sample protein is added between glue glass plate；5) by wire by the first positive electrical pole and the first negative electricity pole with power supply just
Negative pole connects, and is required according to specific experiment, it is determined that suitable power parameter completes separation of the sample protein on gel；Separate
Cheng Hou, closes power supply, takes out gel；6) gel of taking-up is laid in the 3rd folder of preparative gel electrophoresiss eluting collection device
On the medial surface of plate, then will face down on the inside of the 4th clamping plate, cover on three-ply board, and use screw lock；7) respectively to
Electrode buffer is added in each eluting room and the second negative electrode liquid bath, electrode cap is covered, and is closed collecting tank switch；By leading
Second positive electrical pole and the second negative electricity pole are connected by line with the both positive and negative polarity of power supply, select suitable power parameter so that
Albumen in gel is in each eluting room is eluted on gel thicknesses direction；8), after eluting is finished, close power supply simultaneously
Open collecting tank switch so that the solution in each eluting room is had respectively entered in the collecting tank of lower section, will be collected using liquid-transfering gun
Solution in groove completes to collect by being drawn in sample cell, to treat subsequent analysis and use.
Due to taking above technical scheme, which has advantages below to the present invention：1st, the present invention is due to preparative gel electrophoresiss
In segregation apparatuss, respectively positioned at the upper and lower of gel, positive and negative electrode buffer is vertical with gel formation electric for positive and negative electrode liquid bath
Swimming, it is short the time required to separating, with higher separation efficiency.2nd, the present invention is due in preparative gel electrophoresiss eluting collection device
Negative electrode liquid bath and each eluting room respectively positioned at gel both sides, during collection be based on the electroelution on gel thicknesses direction come
Complete, due to gel thicknesses be far smaller than gel length and width, therefore the present invention collect when the time required to it is short, compare tradition
Prepare electrophoresis more efficient.3rd, it is of the invention as two clamping plates of preparative gel electrophoresis separating device adopt rectangle structure, make
Two cooling pieces of device for cooling are located on the outside of two clamping plates respectively, and gel is radiated, compact, and perfect heat-dissipating.
4th, the present invention is simple to operate, and experimenter need to only complete with glue, loading, take glue, adds the operation that eluent and power supply are arranged
Preparation process is completed, tradition is compared and is prepared electrophoresis needs long-time frequently taking-up eluted product, operate easier and hommization.
5th, the present invention due to be prepare between the two glue glass plates needed for gel, for different samples can unrestricted choice gel match somebody with somebody
System, motility are higher, compare tradition and prepare the gel column that each experiment of electrophoresis is required for commercialization, reduce cost.6th, this
It is bright as each part can be dismantled repeatedly, low cost, it is easy to reuse.Present configuration is simple, easy to operate, Ke Yiguang
It is general to be applied in proteomics research field.
Description of the drawings
Fig. 1 is preparative gel electrophoresis separating device schematic diagram of the present invention
Fig. 2 is preparative gel electrophoresiss eluting collection device schematic diagram of the present invention
Fig. 3 is understructure schematic diagram of the present invention
Fig. 4 is base profile of the present invention
Fig. 5 is a clamping plate schematic diagram in preparative gel electrophoresiss eluting collection device of the present invention
Fig. 6 is another clamping plate schematic diagram in preparative gel electrophoresiss eluting collection device of the present invention
Fig. 7 is electrode cap schematic diagram in preparative gel electrophoresiss eluting collection device of the present invention
Fig. 8 is collecting tank schematic diagram in preparative gel electrophoresiss eluting collection device of the present invention
Fig. 9 is the gel-colored scanning figure after being separated to sample protein using the present invention
Figure 10 is collected after sample protein using the present invention, takes out gel therein the figure for being dyeed
Figure 11 is the gel-colored scanning figure after being collected to the sample protein in Fig. 9 using the present invention
Figure 12 is the gel-colored scanning figure after being separated to sample protein using the present invention
Figure 13 is the gel-colored scanning figure after being collected to the sample protein in Figure 12 using the present invention
Specific embodiment
With reference to the accompanying drawings and examples the present invention is described in detail.
As shown in Figure 1 and Figure 2, micropreparation type gel electrophoresis apparatus of the present invention include preparative gel electrophoresis separating device 1
With preparative gel electrophoresiss eluting collection device 2.Preparative gel electrophoresis separating device 1 complete sample protein on gel point
From preparative gel electrophoresiss eluting collection device 2 carries out eluting collection to sample protein separated on gel.Wherein, prepare
Type gel electrophoresis separating device 1 includes two clamping plates, 11,12, two glue glass plate, 13,14, one base 15 and a refrigerating plant.Two
Glue glass plate 13,14 is separately positioned in the groove that two clamping plates 11,12 medial surface are arranged, and the depth of groove is less than two glues
The thickness of glass plate 13,14.Clamping plate 11,12 medial surface are staggered relatively and by some screw locks, and two clamping plates 11,12 bottoms
The boss of setting is plugged in base 15.Refrigerating plant includes two cooling pieces, is separately positioned on the outside of clamping plate 11 and clamping plate 12,
And it is parallel with the vertical plane of clamping plate 11,12.Preparative gel electrophoresiss eluting collection device 2 includes two clamping plates, 21,22, one electrode cap
23 and a collecting tank 24.The medial surface of two clamping plates 21,22 is staggered relatively and by some screw locks.Electrode cap 23 is arranged on two
The top of clamping plate 21,22, collecting tank 24 are arranged on the lower section of two clamping plates 21,22.
As shown in figure 1, a negative electrode liquid bath 111 is provided with the top of clamping plate 11,111 bottom of negative electrode liquid bath is provided with
Two wire columns 112, are wound with platinum electrode silk 113 on two wire columns 112, and the platinum electrode silk 113 be arranged on 11 upper table of clamping plate
The negative electricity pole 114 in face is connected.The medial surface of negative electrode liquid bath 111 is provided with an opening 115, and 115 periphery of opening is arranged
There is " U " type silicon rubber bar 116, " U " type silicon rubber bar 116 is partially submerged in the medial surface of clamping plate 11, and " U " type silicon rubber bar
116 enclosed areas are slightly larger than opening 115.13 upper end of glue glass plate is also equipped with one with 111 upper shed 115 of negative electrode liquid bath
Corresponding opening 131.
As shown in Figure 3, Figure 4, base 15 is closed box structure, and which is internally provided with two wire columns 151, two wire columns
Platinum electrode silk 152, and the platinum electrode silk 152 and 153 phase of positive electrical pole for being arranged on 15 upper surface of base are wound with 151
Even.The upper surface of base 15 is additionally provided with an opening 154, be open 154 width should meet can place down two clamping plates 11,12 with
And two glue glass plates 13,14.
As shown in figure 5, some eluting rooms 211 at the top of clamping plate 21, have been arranged side by side, and the medial surface of each eluting room 211 is all provided with
It is equipped with an opening 212.Each 211 bottom of eluting room is provided with longitudinal cylindrical channel 213 of straight-through 21 bottom of clamping plate, and
A horizontal cylinder collecting tank switch 214 is additionally provided with below each eluting room 211, is provided with collecting tank switch 214 vertical with each
To the hole of the matching of cylindrical channel 213, for controlling the discharge of liquid in each eluting room 211.It is additionally provided with inside clamping plate 21
One platinum electrode silk 215, platinum electrode silk 215 run through all eluting rooms 211, and be arranged on the positive electrical pole on 21 surface of clamping plate
216 are connected.
As shown in fig. 6, a negative electrode liquid bath 221 is provided with the top of clamping plate 22,221 bottom of negative electrode liquid bath is provided with
One platinum electrode silk 222, and platinum electrode silk 222 is connected with the negative electricity pole 223 for being arranged on 22 surface of clamping plate.Negative electrode liquid bath
It is provided with some openings 224 on 221 medial surface, and each opening 224 is opened with each 211 medial surface of eluting room of clamping plate 21 respectively
Mouth 212 is corresponding.The medial surface of negative electrode liquid bath 221 is additionally provided with " U " type silicon rubber bar 225, " U " type silicon rubber bar
225 are partially submerged in clamping plate 22, and 225 enclosed area of " U " type silicon rubber bar is slightly larger than negative electrode liquid bath 221.
As shown in Figure 7, Figure 8, be provided with electrode cap 23 two respectively with clamping plate 21,22 on positive and negative electrode electrode column
216th, 223 parent part 231 for matching.The groove 241 matched with each eluting room 211 in clamping plate 21 is provided with collecting tank 24,
For being collected to the protein fraction of eluting in each eluting room 211.
In above-described embodiment, 11 bottom of clamping plate in preparative gel electrophoresis separating device 1 is additionally provided with a rectangle hole,
And rectangle hole is located at the lower section of negative electrode liquid bath 111.One is also equipped with clamping plate 12 with the symmetrical rectangular opening of clamping plate 11
Hole, is preferably the gel radiating between two glue glass plates 13,14 for causing refrigerating plant 16.
The negative electrode liquid bath at the top of clamping plate 22 in above-described embodiment, in preparative gel electrophoresiss eluting collection device 2
Some supports 226 are provided with 221, the side for preventing clamping plate 22 is bent in experimentation.
Based on micropreparation type gel electrophoresis apparatus of the present invention, the using method of the present invention is comprised the following steps：
1) first two glue glass plates 13,14 are inserted respectively in clamping plate 11,12 corresponding grooves, successively will with screw
Clamping plate 11, two glue glass plates 13,14 and clamping plate 12 are locked.Wherein, " U " type silicon rubber bar 116 is provided for glue glass plate 13
Buffering.
2) the corresponding solution of desired concn is prepared, and is poured into two pieces of glues of preparative gel electrophoresis separating device 1
In glass plate 13,14, the gel for forming solid-state in a moment is stood.
3) add electrode buffer in from the opening 154 on 15 surface of base to base 15, and by clamping plate 11, two glue glass
In 12 insertion opening 154 of plate 13,14 and clamping plate.
4) electrode buffer is added in negative electrode liquid bath 111, on electrode buffer is by glue glass plate 13
The opening of side is immersed between two glue glass plates 13,14, " U " type silicon rubber bar 116 can prevent negative electrode liquid bath 111 with
Leakage between glue glass plate 13.Afterwards, add sample protein between two glue glass plates 13,14, due to sample protein it is close
Degree is larger, its can by electrode buffer drop to loading groove above gel (i.e. gel prepare after formed above gel it is recessed
Groove) in.
5) respectively positive and negative electrode electrode column 153,114 is connected with the both positive and negative polarity of power supply by wire, will according to specific experiment
Ask, it is determined that suitable power parameter completes separation of the sample protein on gel, now, the sample protein on gel can according to point
Son amount size separation becomes band (as shown in Figure 9).Power supply is closed, and is turned on screw and two glue glass is taken out from two clamping plates 11,12
Plate 13,14, and gel is taken out from two glue glass plates 13,14.
6) gel of taking-up is carefully laid in the medial surface of the clamping plate 22 of preparative gel electrophoresiss eluting collection device 2
On, then the inner side of clamping plate 21 is faced down, is covered on the medial surface of clamping plate 22, and locked with screw 25." U " type silicon rubber bar
225 are used to provide buffering for gel, neither make gel occur deforming by a relatively large margin, gel is deposited between clamping plate 21,22
In too big space, while preventing leakage.
7), after electrode buffer being added into each eluting room 211 and negative electrode liquid bath 221 respectively, cover electrode cap 23,
And guarantee that collecting tank switch 214 is closed.By wire respectively by positive and negative electrode electrode column 216,223 with power supply just
Negative pole connects, and selects suitable power parameter, such as voltage 50V-70V, electric current 10mA-50mA so that albumen is perpendicular to gel
It is eluted on thickness direction in each eluting room 211.
8) after eluting is finished, close power supply and open collecting tank switch 214 so that the solution difference in each eluting room 211
Below entering in corresponding collecting tank 24, complete to receive by be drawn in sample cell by the solution in collecting tank 24
Collection, to treat subsequent analysis and use.
Preparative gel electrophoresis separating device of the present invention 1 can realize separation of the biological sample on gel.Work as filling
When the gel of note is PAGE gel, then the albumen in sample protein can be distributed in SDS-PAGE by its molecular weight is descending
The negative pole end of gel is to positive terminal；When the gel of perfusion is non-denaturing polyacrylamide (Native-PAGE) gel, then sample
In albumen electrically charged can be separated according to their molecular size range and institute.When sample protein is separated on corresponding gel
Afterwards, place it in preparative gel electrophoresiss eluting collection device 2.Under the conditions of certain voltage and current, in gel
Albumen can enter corresponding molecular weight of albumen area from gel thicknesses direction, according to the corresponding different protein bands of respective molecular weight
Between the eluting room 211 that is located, realize eluting and the collection to different protein fractions.Protein fraction after collection can carry out mass spectrum,
Liquid chromatographic detection meets other and prepares needs.With reference to specific embodiment, the present invention is described further.
Embodiment 1
Using the present invention to bovine serum albumin, beta-casein, horse heart myoglobin and alpha-lactalbumin mixture (below
Abbreviation sample protein) carry out separating and eluting collection, and its performance is studied.Wherein, the preparation method of sample protein is：
Each 500 μ g of bovine serum albumin, beta-casein, horse heart myoglobin and alpha-lactalbumin or so are weighed, after mixing, is added pure
Water, 5 × SDS-PAGE sample-loading buffers (250mM trishydroxymethylaminomethane hydrochloric acid (Tris-HCl) (pH=6.8), 10% (w/
V) SDS, 0.5% (w/v) bromophenol blue, 50% (v/v) glycerol) and beta -mercaptoethanol, it is configured to the standard protein mixing of 10 μ g/ μ L
Thing solution, wherein the beta -mercaptoethanol containing 1 × SDS-PAGE sample-loading buffers and 1%, 10 points of heat denatured in boiling water bath
Clock, be placed in -20 DEG C it is standby.
As shown in figure 9, being separated to sample protein using preparative gel electrophoresis separating device.12% is prepared respectively
Polyacrylamide gel separation gel and 4% polyacrylamide gel concentration glue, poured between two glue glass plates 13.Treat
After gel sets, electrode buffer is added in anode electrode liquid bath 141 and negative electrode liquid bath 111 respectively, in loading groove
Add the standard protein mixture solution of more than 15 μ L (150 μ g).Switch on power, rotational voltage is 90V-160V, to sample egg
Separated in vain.Stop dividing when blue bromophenol blue band moves adjacent to the lower edge of preparative gel electrophoresis separating device
From.Gel is taken out, coomassie brilliant blue staining is used.It can be seen that present invention achieves efficiently dividing to 4 kinds of standard proteins
From, and have good separating effect Jing after test of many times, illustrate that the present invention has repeatability well.
As shown in Figure 10, washed using 2 pairs of gels isolated of preparative gel electrophoresiss eluting collection device of the present invention
It is de- to collect, and the gel after eluting collection is dyeed with Coomassie brilliant blue.It can be seen that collect after gel in
In addition to 24 edge of collecting tank and collecting tank 24 are spaced corresponding position, remainder illustrates the present invention without protein residue
The purpose of complete eluting can be reached.
As shown in figure 11, the sample in the sample cell collected is carried out into lyophilizing concentration, and carries out SDS-PAGE experiments.Can
To find out, the protein band of the sample of collection on SDS-PAGE presses molecular size range arrangement, illustrates that the present invention can be according to
Protein molecular weight is of different sizes to be separated to which.
Embodiment 2
As shown in figure 12, using preparative gel electrophoresis separating device of the present invention 1 to Bacillus subtilis LM 4-
In 2 fungal cells, protein is separated.Using the broken Bacillus subtilis LM 4-2 fungal cells of Ultrasound Instrument, extract
Funguses whole protein, protein concentration is concentrated into after 10-20 μ g/ μ L, thermal denaturation is carried out to albumen, then in preparative gel electrophoresiss
Loading in segregation apparatuss, method is with embodiment 1.As can be seen that the present invention can be according to protein molecular weight opposite of different sizes
Thing actual sample is separated.
As shown in figure 13, washed using 2 pairs of gels isolated of preparative gel electrophoresiss eluting collection device of the present invention
De- and collection.As can be seen that protein band of the funguses whole protein each component on SDS-PAGE presses molecular size range arrangement, explanation
The present invention can reach the purpose that high efficiency separation is carried out to protein in complex biological sample.
Embodiment 3
Sample protein is separated using the present invention and eluting is collected, and calculated its response rate.Ox blood serum is weighed first
55 μ g of albumin (BSA), add pure water, 5 × SDS-PAGE sample-loading buffers (250mMTris-HCl (pH=6.8), 10%
(w/v) SDS, 0.5% (w/v) bromophenol blue, 50% (v/v) glycerol) and beta -mercaptoethanol, the standard protein for being configured to 5 μ g/ μ L is molten
Liquid, wherein the beta -mercaptoethanol containing 1 × SDS-PAGE sample-loading buffers and 1%.Standard protein solution is added in boiling water bath
After thermal denaturation 10 minutes, preparative gel electrophoresis separating device 1 and preparative gel electrophoresiss eluting collection device 2 pairs is respectively adopted
Which carries out separating and eluting is collected.After the completion of collection, by each component lyophilizing weight molten to 200 μ L.
Then, each 200 μ L of BSA aqueous solutions that normal concentration is 0.1 μ g/ μ L and 0.5 μ g/ μ L are prepared, respectively in liquid phase color
Loading (chromatographic column in spectrum：C18 reversed phase chromatographic column；Mobile phase A：98%H2O, 2%ACN；Mobile phase B：98%ACN, 2%H2O；0-
20min, -95% Mobile phase B of 5% Mobile phase B；20-30min, 95% Mobile phase B).Observation sample appearance time in the liquid phase
And peak area, obtain：The BSA aqueous solutions appearance time of 0.1 μ g/ μ L is 12.8-13.2min, and peak area is 419.98；0.5μg/
The BSA aqueous solutions appearance time of μ L is 12.8-13.2min, and peak area is 2428.42.Afterwards, by each component (group collected
Point 1- components 15) loading in liquid chromatograph successively, observes each component and goes out peak area in 12.8-13.2min, using sandwich
Method, goes out the BSA total amounts collected, can obtain of the invention according to the calculated by peak area of BSA loadings in liquid chromatograph of normal concentration
The response rate be 70.9%.
The various embodiments described above are merely to illustrate the present invention, wherein the structure of each part, connected mode and processing technology etc. are all
Can be what is be varied from, every equivalents carried out on the basis of technical solution of the present invention and improvement should not be excluded
Outside protection scope of the present invention.
Claims (6)
1. a kind of micropreparation type gel electrophoresis apparatus, it is characterised in that：It includes preparative gel electrophoresis separating device and system
Standby type gel electrophoresiss eluting collection device；The preparative gel electrophoresis separating device include first clamping plate, second clamping plate, first
Glue glass plate, the second glue glass plate, a base and a refrigerating plant；The first glue glass plate and the second glue glass
Plate is separately positioned in the groove that the first clamping plate and second clamping plate medial surface are arranged, and the depth of groove described in two is respectively less than
The thickness of the first glue glass plate and the second glue glass plate；The first clamping plate and second clamping plate medial surface are staggered relatively
And be plugged in the base by some screw locks, and the boss that the first clamping plate and second clamping plate bottom are arranged；Institute
Stating refrigerating plant includes two cooling pieces, and cooling piece described in two is separately positioned on the outside of the first clamping plate and second clamping plate, and
It is parallel with the vertical plane of the first clamping plate and second clamping plate；The preparative gel electrophoresiss eluting collection device includes the 3rd folder
Plate, the 4th clamping plate, an electrode cap and a collecting tank；If the medial surface of the three-ply board and the 4th clamping plate is staggered relatively and passes through
Dry screw lock；The electrode cap is arranged on the top of the three-ply board and the 4th clamping plate, and the collecting tank is arranged on described
The lower section of three-ply board and the 4th clamping plate；
The first negative electrode liquid bath is provided with the top of the first clamping plate, at the top of the 4th clamping plate, the second negative electrode is provided with
Liquid bath；Some eluting rooms are arranged side by side at the top of the three-ply board, the medial surface of each eluting room is provided with the 4th and opens
Mouthful；A longitudinal cylindrical channel for leading directly to the three-ply board bottom is provided with below each eluting room；And it is each described
A laterally cylindrical collecting tank switch is additionally provided with below eluting room, is provided with and each longitudinal direction on the collecting tank switch
The hole of cylindrical channel matching, for controlling the discharge of each eluting indoor liquid；It is additionally provided with the three-ply board
3rd platinum electrode silk, the 3rd platinum electrode silk run through all eluting rooms, and be arranged on the three-ply board surface
Second positive electrical pole is connected.
2. a kind of micropreparation type gel electrophoresis apparatus as claimed in claim 1, it is characterised in that：First negative electrode
Liquid bath bottom is provided with two first wire columns, and the first platinum electrode silk, and first platinum are wound with the first wire column described in two
Wire electrode is connected with the first negative electricity pole for being arranged on the first clamping plate upper surface；The first negative electrode liquid bath it is interior
Side is provided with the first opening, and first mouth periphery is provided with first " U " type silicon rubber bar；First " U " the type silicon rubber
Adhesive tape is partially submerged in the medial surface of the first clamping plate, and the enclosed area of first " U " the type silicon rubber bar is slightly larger than described
First opening；The first glue glass plate upper end be provided with one with the first negative electrode liquid bath it is described first opening phase
The second opening answered.
3. a kind of micropreparation type gel electrophoresis apparatus as claimed in claim 1, it is characterised in that：The base is fully sheathed case
Body structure, which is internally provided with two second wire columns, and the second platinum electrode silk, and described are wound with the second wire column described in two
Two platinum electrode silks are connected with the first positive electrical pole for being arranged on the base upper surface；The upper surface of the base is additionally provided with
3rd opening, the width of the 3rd opening should meet and can put down the first clamping plate, second clamping plate, the first glue glass plate
With the second glue glass plate.
4. a kind of micropreparation type gel electrophoresis apparatus as claimed in claim 2, it is characterised in that：The base is fully sheathed case
Body structure, which is internally provided with two second wire columns, and the second platinum electrode silk, and described are wound with the second wire column described in two
Two platinum electrode silks are connected with the first positive electrical pole for being arranged on the base upper surface；The upper surface of the base is additionally provided with
3rd opening, the width of the 3rd opening should meet and can put down the first clamping plate, second clamping plate, the first glue glass plate
With the second glue glass plate.
5. a kind of micropreparation type gel electrophoresis apparatus as described in any one of Claims 1-4, it is characterised in that：Described
Two negative electrode liquid bath bottoms are provided with the 4th platinum electrode silk, and the 4th platinum electrode silk be arranged on the 4th clamping plate table
The second negative electricity pole in face is connected；It is provided with some 5th openings on the medial surface of the second negative electrode liquid bath, and respectively
5th opening is corresponding with the 4th opening of each described eluting indoor profile of the three-ply board respectively；Described second is negative
The medial surface of pole electrode reservoir is additionally provided with second " U " type silicon rubber bar, and second " U " the type silicon rubber bar is partially submerged in institute
The 4th clamping plate medial surface is stated, and the enclosed area of second " U " the type silicon rubber bar is slightly larger than the second negative electrode liquid bath；Institute
State be provided with electrode cap two respectively with the three-ply board and the 4th clamping plate on the second positive and negative electrode electrode column phase
The parent part matched somebody with somebody；The groove matched with each eluting room in the three-ply board is provided with the collecting tank, for each
In the eluting room, the protein fraction of eluting is collected.
6. a kind of using method of the micropreparation type gel electrophoresis apparatus as described in any one of Claims 1 to 5, including following step
Suddenly：
1), after first, second glue glass plate being inserted respectively into first clamping plate and the corresponding groove of second clamping plate, locked with screw
Tightly；
2) the corresponding solution of desired concn is prepared, and is poured into the first glue glass plate of preparative gel electrophoresis separating device
And second between glue glass plate, the gel for forming solid-state in a moment is stood；
3) be open from the 3rd of susceptor surface the and electrode buffer added into base, and by first clamping plate, the first glue glass plate,
In the 3rd opening of second glue glass plate and second clamping plate insertion；
4) electrode buffer is added in the first negative electrode liquid bath, until electrode buffer passes through the first glue glass plate
Opening be immersed between the first glue glass plate and the second glue glass plate；To the first glue glass plate and the second glue glass
Sample protein is added between plate；
5) the first positive electrical pole and the first negative electricity pole are connected with the both positive and negative polarity of power supply by wire, according to specific experiment
Require, it is determined that suitable power parameter completes separation of the sample protein on gel；After the completion of separation, power supply is closed, take out solidifying
Glue；
6) gel of taking-up is laid on the medial surface of three-ply board of preparative gel electrophoresiss eluting collection device, then will
Face down on the inside of 4th clamping plate, cover on three-ply board, and use screw lock；
7) electrode buffer is added into each eluting room and the second negative electrode liquid bath respectively, cover electrode cap, and close collection
Groove is switched；The second positive electrical pole and the second negative electricity pole are connected with the both positive and negative polarity of power supply by wire, select suitable
Power parameter so that the albumen in gel is in each eluting room is eluted on gel thicknesses direction；
8), after eluting is finished, close power supply and open collecting tank switch so that the solution in each eluting room has respectively entered lower section
Collecting tank in, complete by be drawn in sample cell by the solution in collecting tank collect, with treat subsequent analysis and
Use.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018067736A1 (en) * | 2016-10-04 | 2018-04-12 | Sage Science, Inc. | Apparatuses, methods and systems for automated processing of nucleic acids and electrophoretic sample preparation |
| CN108459068B (en) * | 2018-06-14 | 2024-04-30 | 沧州医学高等专科学校 | Automatic device for gel preparation and use method |
| CN112206659A (en) * | 2019-07-12 | 2021-01-12 | 华大青兰生物科技(无锡)有限公司 | Integrated polyacrylamide gel electrophoresis device |
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