CN106749504B - Horizontal column-shaped gel electrophoresis device for protein separation - Google Patents
Horizontal column-shaped gel electrophoresis device for protein separation Download PDFInfo
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- CN106749504B CN106749504B CN201611101473.6A CN201611101473A CN106749504B CN 106749504 B CN106749504 B CN 106749504B CN 201611101473 A CN201611101473 A CN 201611101473A CN 106749504 B CN106749504 B CN 106749504B
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Abstract
A horizontal cylindrical gel electrophoresis device for protein separation, the horizontal cylindrical gel electrophoresis device employing a horizontal configuration, comprising: a cylindrical gel tube filled with gel for protein sample separation; the length of the columnar gel tube is 8-12cm, and 8cm is preferred; the electrophoresis tank comprises 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 tank on the positive side; the electrophoresis tank is provided with a position for placing the columnar gel tube. The column-shaped gel electrophoresis device can be used for off-line separation and on-line separation and detection of proteins with different molecular weights according to different filled polyacrylamides and by combining with instruments such as ICP-MS and the like, has the characteristics of short analysis time, low cost, simple manufacturing process, small dead volume, wide selection range of separation conditions, high separation degree, capability of being modified according to actual requirements and the like, and has the possibility of continuous optimization.
Description
Technical Field
The invention belongs to the technical field of biochemical detection and analysis, and particularly relates to a horizontal cylindrical gel electrophoresis device for protein separation.
Background
Proteins are basic constituent substances of living bodies, are executives of various important functions of living bodies, and have important research significance and practical value on the research of protein composition, structure and function. Among them, the electrophoresis technique is the most widely and effectively used method in protein separation and determination.
The electrophoresis technology mainly comprises isoelectric focusing electrophoresis (IEF) and polyacrylamide gel electrophoresis (PAGE), wherein the PAGE takes polyacrylamide gel with a net-shaped three-dimensional structure as a supporting medium, and under the action of an electric field, protein molecules to be separated are separated into a plurality of obvious zones according to different molecular charge, sizes and shapes. PAGE can be further classified into denaturing (SDS-PAGE) and non-denaturing gel electrophoresis (Native-PAGE). Denaturing gel electrophoresis (SDS-PAGE) inactivates proteins by reacting Sodium Dodecyl Sulfate (SDS) with the proteins, and can convert the proteins into substances with different molecular weights and similar structures, so that the proteins can be separated according to the difference of the molecular weights regardless of the properties of the proteins, and the proteins with small mass-to-charge ratios move fast and far, and the proteins with large mass-to-charge ratios move slowly. SDS-PAGE is easy and flexible to operate, high in resolution and good in reproducibility, and is the most widely used protein separation technology.
Depending on the device form of the support, gel electrophoresis can be further classified into flat plate electrophoresis, vertical plate electrophoresis and column electrophoresis. Wherein the columnar gel is more beneficial to the concentration of the sample and the collection after separation and the combination with a subsequent detection system. The column gel electrophoresis can select glass tubes with different inner diameters and lengths according to requirements, agarose filled in the glass tubes can be used for separating nucleic acid, and polyacrylamide gel filled in the glass tubes can be used for separating protein. And eluting the protein subjected to the column electrophoretic separation by using an elution device, and analyzing and detecting the protein by combining with an inductively coupled plasma mass spectrometer (ICP-MS) technology. The combination of column gel electrophoresis and ICP-MS technology has been successfully applied to the on-line separation of DNA, RNA and small molecular substances.
However, although the existing commercial column-shaped gel electrophoresis device can be used for separating and detecting various substances such as polypeptide, RNA, DNA, protein and the like, and has good universality, the inner diameter of the gel column is larger, and the dead volume of the elution device is larger, so that the whole column-shaped gel electrophoresis device has lower separation efficiency and analysis sensitivity to the protein, and has high manufacturing cost and complicated operation. How to optimize and improve the column electrophoresis apparatus is an important factor affecting protein analysis and detection.
The 3D printing technique is a technique that is a distinct inverse of the traditional material processing method, and is based on three-dimensional CAD model data to rapidly construct objects by adding material to the layer-by-layer fabrication. The printing process is realized by a digital technical material printer by adopting a manufacturing method of directly manufacturing a three-dimensional physical solid model completely consistent with a corresponding mathematical model. The technology is characterized in that the technology can manufacture articles with almost any shape, and the complex process of a plurality of processing procedures required by the traditional technology is eliminated, so that the manufacturing period is short and the cost is low. In recent years, 3D printing technology has been rapidly developed as a leading-edge advanced manufacturing technology, and has gradually played an important role in the fields of industrial manufacturing, biomedicine, building manufacturing, cultural art, and the like. However, there is no precedent to apply 3D printing technology to a cylindrical gel electrophoresis device.
Disclosure of Invention
In view of the above, the main objective of the present invention is to provide a horizontal column gel electrophoresis apparatus for protein separation, which solves at least one of the above-mentioned problems.
To achieve the above object, as one aspect of the present invention, there is provided a horizontal column gel electrophoresis apparatus for protein separation, the horizontal column gel electrophoresis apparatus employing a horizontal structure, comprising:
a cylindrical gel tube filled with gel for protein sample separation; the length of the columnar gel tube is 8-12cm, and 8cm is preferred; and
an electrophoresis tank including a buffer solution tank on a positive side, a buffer solution tank on a negative side, a cooling water tank located in the middle, and an elution solution tank located on the positive side; the electrophoresis tank is provided with a position for placing the columnar gel tube.
Wherein, the outer diameter of the columnar gel tube is 4-6mm, preferably 4 mm; the inner diameter of the cylindrical gel tube is 2-2.2mm, preferably 2.2 mm.
Wherein the electrophoresis tank is manufactured by a 3D printing technology;
preferably, the electrophoresis tank is made of high molecular polymer material, preferably PLA plastic.
Wherein the depth of the electrophoresis tank is 3.5-5cm, the width is 8-12cm, and the total length is 18-22 cm;
preferably, the length of the cooling water tank in the electrophoresis tank is 5.5-9cm, preferably 5.5 cm; the lengths of the buffer solution tank and the elution solution tank on the positive electrode side are 5-6cm, and 6cm is preferred; the length of the buffer solution tank on the negative electrode side is 5 to 6cm, preferably 5 cm.
The wall of the buffer solution tank on the positive electrode side adjacent to the wall of the elution solution tank is provided with an elution tube jack, and the outside of the same side of the buffer solution tank on the positive electrode side and the buffer solution tank on the negative electrode side is respectively provided with an electrode seat of a positive electrode and an electrode seat of a negative electrode and provided with electrodes;
preferably, a movable clamping plate is arranged at the top end of the outer part of one side of the cooling water tank and used for fixing the columnar gel pipe and sealing the cooling water tank; and two screwing screw holes are respectively formed in one end of the movable clamping plate and the other side of the cooling water tank, and the movable clamping plate is screwed by inserting screws during use.
The horizontal columnar gel electrophoresis device also comprises an elution device, wherein the elution device comprises a front part and a rear part which can be assembled;
preferably, the front end of the front half part of the elution device is provided with a jack of a cylindrical gel tube with the length of 6-8mm and the diameter of 5.5-7.5 mm; the middle of the front half part is a round hole with the length of 1.3mm and the diameter of 4mm, and is used for placing a first SPE sieve plate, and two sides of the round hole are respectively connected through an elution pipe; the rear end of the front half part is provided with a port with the length of 4.1mm and the diameter of 11.2mm, and the port is connected with the rear half part; a round hole with the length of 1.6mm and the diameter of 9.2mm is formed in the front end of the rear half part of the elution device and used for placing a second SPE sieve plate, and a dialysis membrane is placed at the front end of the second SPE sieve plate; the rear end of the rear half part is provided with a connecting hole communicated with the buffer solution storage container.
Wherein, the main body part of the elution device is made of high molecular polymer material, preferably synthetic resin;
preferably, the elution device body portion is manufactured by 3D printing techniques.
Wherein, in use, a cylindrical gel tube is inserted into the first half of the elution apparatus and contacts the first SPE sieve deck;
preferably, when the elution device is used, the front part and the rear part are assembled together, and the sealing film is tightly wound on the joint.
Wherein, it is right before the elution device uses need first SPE sieve, second SPE sieve and dialysis membrane fully wet, and the air is removed in order to guarantee the effect to the side.
Wherein, when the elution device is used, the elution pipe should be arranged at the upper end and the lower end, the upper end elution pipe is inserted into the elution solution tank through the insertion hole of the elution pipe on the electrophoresis tank, the elution pipe at the lower end is connected with the sleeve on the peristaltic pump side, the eluent enters from the upper end elution pipe, the elution device is led out by the lower end elution pipe after passing through the first SPE sieve plate, and the inductively coupled plasma mass spectrometer is introduced under the flow rate control of the peristaltic pump.
Based on the scheme, the horizontal cylindrical gel electrophoresis device has the following beneficial effects: (1) the horizontal column-shaped gel electrophoresis device can be used for the rapid off-line separation of protein; (2) the horizontal column-shaped gel electrophoresis device can realize rapid and accurate on-line separation and detection of protein samples by combining with analytical instruments such as ICP-MS and the like, and improves the separation efficiency and the analysis sensitivity of the protein; (3) the column-shaped gel tube can realize the rapid separation of different proteins by matching with an electrophoresis tank according to the difference of filled polyacrylamide gel; (4) the main body of the horizontal columnar gel electrophoresis device is manufactured by 3D printing, the used materials are about 300g, the manufacturing cost is about 500 yuan RMB, the whole manufacturing process is less than 18 hours, the weight is light, the cost is low, and the manufacturing process is rapid, simple and convenient; (5) the elution device has small dead volume and low price, and can be used for one time or repeatedly; (6) the columnar gel electrophoresis device has simple structure and easy operation; (7) the gel electrophoresis device has higher protein separation efficiency, has the characteristics of low cost, simple manufacturing process, small dead volume, rapidness, accuracy and the like, and has the possibility of continuous optimization.
Drawings
FIG. 1 is a schematic structural view of a cylindrical gel tube of the present invention;
FIG. 2 is a schematic diagram of the structure of an electrophoresis tank of the present invention;
FIGS. 3A and 3B are a cross-sectional view of the left half of an elution apparatus of the present invention and an exploded schematic view of the elution apparatus, respectively;
FIGS. 4A and 4B are graphs showing the separation effect of SDS-PAGE provided by Bio-Rad on the separation of Precision Plus Protein Dual Color Protein standards produced by Bio-Rad using the column gel electrophoresis Protein separation apparatus (column gel tube and electrophoresis chamber) of the present invention, respectively;
FIG. 5 shows two types of samples I combined with ICP-MS using the gel electrophoresis protein separation apparatus of the present invention127And (3) an electrophoresis result chart of on-line separation and detection of the marked standard protein, wherein the peak 1 is RA protein, and the peak 2 is CA protein.
FIG. 6A shows the gel electrophoresis protein separation apparatus of the present invention in combination with ICP-MS for three samples I127Fig. 6B is a graph showing the results of detection of the same three standard proteins using an optimized commercial electrophoresis apparatus under the same conditions, wherein peak 1 is RA protein, peak 2 is CA protein, and peak 3 is BSA protein.
In the above figures, the reference numerals have the following meanings:
1. -a buffer solution tank; 2. -an elution solution tank; 3. -a cooling water tank; 4. -a negative electrode; 5. -a positive electrode; 6. -an electrode wire slot; 7. -a gel tube receiving hole; 8. -an elution tube receptacle; 9. -a cooling water inlet; 10. -a cooling water outlet; 11. -a movable card; 12. -tightening the screw hole;
21. -a gel tube receptacle; 22. -a small SPE sieve plate placement hole; 23. -an elution tube; 24. -an interface; 25.- -20. mu. mSPE sieve plate (small SPE sieve plate); 26. -3kD dialysis membrane; 27. -3mm SPE sieve plate (large SPE sieve plate); 28. -large SPE sieve plate placement wells; 29.- -buffer solution communication holes.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The invention relates to a horizontal column-shaped gel electrophoresis device for protein separation, which mainly comprises three components, namely a column-shaped gel tube, an electrophoresis tank and an elution device. Wherein, electrophoresis tank main part and elution device main part adopt 3D to print the manufacturing, and the material is macromolecular material, for example PLA plastics and synthetic resin, and it has following advantage: according to the actual sample condition, the horizontal column-shaped gel electrophoresis device, the column-shaped gel tube and the electrophoresis tank contained in the horizontal column-shaped gel electrophoresis device can be used for off-line separation of protein; the horizontal column-shaped gel electrophoresis device, the column-shaped gel tube, the electrophoresis tank and the elution device contained in the horizontal column-shaped gel electrophoresis device are combined with an analysis instrument such as ICP-MS and the like, and can be used for on-line separation and detection of protein.
When the device is used specifically, when offline detection is needed, different polyacrylamide gels are filled in the cylindrical gel tube, a protein sample to be separated is added into the cylindrical gel tube and placed in the electrophoresis tank, then buffer solution and cooling circulating water are respectively added into different tanks of the gel electrophoresis tank, the movable clamping plate is fastened, an electrode of the electrophoresis tank is powered on, then the protein sample can be separated offline in the gel tube under the action of current, the operation of the whole process is simple and convenient, and the separation efficiency is high.
When on-line detection is needed, filling different polyacrylamide gels in a cylindrical gel tube, placing the cylindrical gel tube in an electrophoresis tank and connecting the cylindrical gel tube with an elution device, connecting one side of the elution device with an injection port of an inductively coupled plasma mass spectrometer through a peristaltic pump, adding a protein sample to be separated into the cylindrical gel tube and placing the protein sample in the electrophoresis tank, then respectively adding a buffer solution, an elution solution and cooling circulating water into different tanks of the gel electrophoresis tank, fastening a movable clamping plate, switching on an electrode of the electrophoresis tank, separating the protein sample in the gel tube under the action of current, and feeding the protein sample into an ICP-MS (inductively coupled plasma-mass spectrometer) to analyze and detect under the action of the peristaltic pump along with an eluent. Therefore, the rapid and accurate online separation and detection of the protein sample can be realized simultaneously, and the whole device is simple and convenient to operate, small in dead volume and high in separation and detection efficiency.
Specifically, the horizontal cylindrical gel electrophoresis device of the present invention comprises a cylindrical gel tube, an electrophoresis tank, and an optional elution device.
The horizontal columnar gel electrophoresis device adopts a horizontal structure, namely, a columnar gel tube is horizontally placed. Different kinds of polyacrylamide gel can be filled in the columnar gel tube, and the corresponding gel is selected according to the detection requirement. For example, if it is desired to isolate high molecular weight proteins, a low concentration gel is filled; if the low molecular weight protein needs to be separated, filling high-concentration gel; if it is desired to separate proteins of broad molecular weight, the gradient gel may be filled. Through repeated experimental verification and calculation, the length of the cylindrical gel tube can be set according to the filling amount of the gel, for example, the length is 8-12cm, the outer diameter is preferably 4-6mm, and is further preferably 4mm, and the inner diameter is preferably 2-2.2mm, and is further preferably 2.2mm, so that the separation efficiency of the protein is greatly improved.
The electrophoresis tank comprises a buffer solution tank at the positive electrode side and a buffer solution tank at the negative electrode side, a cooling water tank positioned in the middle, and an elution solution tank at the positive electrode side. The utility model discloses a positive pole side's buffer solution groove is equipped with elution pipe jack on the wall adjacent with elution solution groove, the outside of two buffer solution groove one sides has the electrode holder of positive negative electrode respectively, and be equipped with the electrode, the electrically conductive platinum silk of being connected with the electrode extends to two buffer solution tank bottoms portion along the wire electrode draw-in groove through the aperture on the groove, there is the gel pipe respectively to lay the hole on the wall that two buffer solution grooves and cooling water tank meet, the both sides of cooling water tank are equipped with cooling water inlet and cooling water outlet respectively, there is movable cardboard on the outside top of cooling water tank one side, be used for fixed gel pipe and closed cooling water tank, activity cardboard one end and cooling water tank opposite side have two screws up screw hole respectively, be used for inserting the screw during use and screw up movable.
Preferably, the electrophoresis tank is made of a high molecular polymer material, such as PLA plastic. Further preferably, the electrophoresis tank is manufactured by 3D printing technology. The depth of the electrophoresis tank is, for example, 3.5 to 5cm, the width is, for example, 8 to 12cm, and the total length is, for example, 18 to 22 cm.
Wherein, the length of the cooling water tank in the electrophoresis tank is, for example, 5.5-9cm, preferably 5.5 cm; the lengths of the positive electrode side buffer solution tank and the elution solution tank are, for example, 5 to 6cm, preferably 6 cm; the length of the negative electrode side buffer solution tank is, for example, 5 to 6cm, preferably 5 cm.
Preferably, the diameter of the gel tube receiving hole on the wall of the buffer solution tank in the electrophoresis tank is, for example, 4 mm.
Wherein, use the thread seal tape winding in order to avoid leaking between the electrophoresis cell wall of the activity cardboard on the outside top of cooling trough one side in the electrophoresis cell and the electrophoresis cell wall that corresponds, insert respectively with the screw during the use in order to screw up the activity cardboard in two screw holes of screwing up of activity cardboard one end and cooling trough opposite side.
Elution device is including two parts in the front and back that can assemble, the front end of first half is provided with long 6-8mm, the diameter is the jack of the column gel pipe of 5.5-7.5mm, the centre adds there is the rubber hose in order to guarantee the gas tightness, the centre of first half is long 1.3mm, the diameter is 4 mm's round hole, a 20 mu m SPE sieve (little SPE sieve) for placing the production of Agilent company, connect through an elution pipe respectively in the both sides of round hole, be provided with long 4.1mm in the rear end of first half, diameter 11.2 mm's interface meets with latter half. The front end of the rear half part is provided with a round hole with the length of 1.6mm and the diameter of 9.2mm, and is used for placing a 3mm SPE sieve plate (a large SPE sieve plate) produced by Aijier company, a 3kD dialysis membrane produced by MYM biotechnology limited company is placed at the front end of the large SPE sieve plate, and a connecting hole communicated with the buffer solution storage container is formed in the rear end of the rear half part.
Preferably, the main body of the elution device is made of, for example, a high molecular polymer material such as a synthetic resin. Further preferably, the elution device body portion is manufactured by 3D printing techniques.
In a preferred embodiment, the elution device is in use, the gel tube of the invention is inserted into the front half of the elution device and contacts the small SPE frit. In another preferred embodiment, the elution device is used by assembling the front and back parts together and wrapping the interface with a sealing film.
Preferably, the elution device needs to fully wet the large SPE sieve plate, the small SPE sieve plate and the dialysis membrane before use, and air is removed to ensure the effect.
Preferably, when the elution device is used, two elution tubes are respectively positioned at the upper end and the lower end, the elution tube at the upper end is inserted into the elution solution tank through an elution tube jack on the electrophoresis tank, the elution tube at the lower end is connected with a sleeve on the peristaltic pump side, and the inductively coupled plasma mass spectrometer is introduced under the flow rate control of the peristaltic pump.
When the horizontal cylindrical gel electrophoresis device is used for off-line separation of protein, two parts, namely a cylindrical gel tube and an electrophoresis tank, are required to be used simultaneously; when the horizontal column-shaped gel electrophoresis device is used for the on-line separation of protein, three parts of a column-shaped gel tube, an electrophoresis tank and an elution device are required to be used simultaneously.
The invention also discloses a gel electrophoresis protein separation detection system adopting the horizontal cylindrical gel electrophoresis device.
The invention discloses a horizontal cylindrical gel electrophoresis device for protein separation, which comprises three parts, namely a cylindrical gel tube, an electrophoresis tank and an elution device, wherein the cylindrical gel tube is made by processing quartz glass, and the electrophoresis tank body and the elution device body are printed by a commercial 3D printer. The electrophoresis tank main body is printed by a CubePro 3D desktop printer produced by 3D systems company, the printing material is PLA plastic, the consumption material is about 300g, and the time is about 18 hours. The elution device main body was printed using a Form2 desktop-level 3D printer manufactured by Formlabs corporation, the printing material was synthetic resin, and the consumable consumed about 5g, which took about 1 hour.
Another preferred embodiment of the present invention will be further described with reference to the accompanying drawings.
As shown in FIG. 1, the cylindrical gel tube for protein separation has a length of 8cm, an outer diameter of 4mm and an inner diameter of 2.2 mm.
When the column-shaped gel tube works, polyacrylamide gel with different volumes and concentrations needs to be filled in the column-shaped gel tube, then the column-shaped gel tube is placed on a gel tube placing hole on the electrophoresis tank, the column-shaped gel tube is used in combination with the electrophoresis tank, an elution device and the like, and after a protein sample is added and electrified, the protein sample can move from a negative electrode to a positive electrode under the action of current and is separated.
As shown in FIG. 2, the column-shaped gel electrophoresis tank for protein separation is made of PLA plastic and has a total weight of about 300g, a depth of 4cm, a width of 10cm and a total length of 20 cm. The electrophoresis tank includes: two buffer solution tanks 1 on the positive side and the negative side, an elution solution tank 2, a cooling water tank 3, a negative electrode 4, a positive electrode 5, an electrode wire tank 6, a gel tube placing hole 7, an elution tube jack 8, a cooling water inlet 9, a cooling water outlet 10, a movable clamping plate 11 and a screwing screw hole 12. The electrophoresis tank comprises four tanks, namely a positive side buffer solution tank 1, a negative side buffer solution tank 1, a middle cooling water tank 3 and a positive side elution solution tank 2, wherein the length of the cooling water tank 3 is 5.5cm, the lengths of the positive side buffer solution tank 1 and the positive side elution solution tank 2 are 6cm, and the length of the negative side buffer solution tank 1 is 5 cm. The outside of one side of the two buffer solution tanks is respectively provided with a positive electrode 4 and a negative electrode 5, one side of the buffer solution tank 1 of the positive side and the negative side adjacent to the cooling water tank 3 is respectively provided with a gel tube placing hole 7, the diameter of the gel tube placing hole is 4mm, conductive platinum wires connected with the negative electrode 4 and the positive electrode 5 are respectively laid at the bottom of the buffer solution tank 1 of the positive side and the negative side along an electrode wire tank 6, and the wall of the buffer solution tank 1 of the positive side adjacent to the elution solution tank 2 is provided with an elution tube jack 8. The both sides of cooling trough 3 are equipped with cooling water entry 9 and cooling water export 10, and there is movable cardboard 11 cooling trough one side for fixed gel pipe and closed cooling trough 3, and 11 one end of movable cardboard and 3 opposite sides of cooling trough have two screw holes 12 of screwing up respectively, are used for screwing up movable cardboard 11.
This electrophoresis tank is at the during operation, buffer about 150ml is added respectively in two buffer solution tank 1 on positive, negative pole side, with long 8cm, the external diameter is 4 mm's column gel pipe and lays the gel pipe and lay hole 7, then buckle movable cardboard 11, it is fixed through screwing up screw hole 12 with two screws, wherein twine the thread seal area between movable cardboard and the electrophoresis tank in order to avoid the weeping, on receiving the power with the electric lead negative electrode 4 and positive electrode 5 respectively, add proper amount elution solution in elution solution tank 2, the elution device one end of cooperation use.
As shown in figures 3A and 3B, the elution device for protein separation comprises a front part and a rear part which can be assembled, wherein the front end of the front half is provided with a jack 21 of a cylindrical gel tube with the length of 8mm and the diameter of 5.6mm, a rubber hose is added in the middle to ensure air tightness, the middle of the front half is provided with a round hole 22 with the length of 1.3mm and the diameter of 4mm and used for placing a 20 mu mSPE sieve plate (small SPE sieve plate) 25 produced by Agilent company, two sides of the round hole are respectively connected with an elution tube, and the rear end of the front half is provided with a connector 24 with the length of 4.1mm and the diameter of 11.2mm and connected with the rear half. The front end of the back half part is provided with a round hole 28 with the length of 1.6mm and the diameter of 9.2mm, and is used for placing a 3mm SPE sieve plate (large SPE sieve plate) 27 produced by Aijier company, the front end of the large sieve plate is provided with a 3kD dialysis membrane 26 produced by MYM biotechnology limited company, and the back end of the back half part is provided with a connecting hole 29 communicated with a buffer solution storage container.
When the elution device works, firstly, a 20-micron SPE sieve plate (small SPE sieve plate) 25 produced by Agilent is placed in a round hole 22, a 3-mm SPE sieve plate (large SPE sieve plate) 27 produced by Aijiel is placed in a round hole 28, a 3kD dialysis membrane 26 produced by MYM biotechnology limited is placed at the front end of the large SPE sieve plate 27, an interface 24 of the front half part is spliced with the rear half part, a sealing membrane is used for tightly winding the spliced part, elution tubes 23 are arranged at the upper end and the lower end, an upper end elution tube 23 is inserted into an elution solution tank 2 through an elution tube jack 8 on an electrophoresis tank, an elution tube 23 at the lower end is connected with a sleeve on the peristaltic pump side, elution enters from the upper end elution tube 23, is led out of the elution device through the lower end elution tube 23 after passing through the small SPE sieve plate 25, and ICP-MS is led in under the flow rate control of the peristaltic pump.
The beneficial effects of the technical scheme of the invention are illustrated by several specific detection embodiments.
Example 1
The horizontal column gel electrophoresis device is used for off-line separation of the protein standard substance and is compared with a commercialized reference result.
The fillers in the cylindrical gel tube were 50. mu.l of 4% concentrated gel and 400. mu.l of 10% separation gel, and the Protein standard added was Precision Plus Protein Dual Color Protein standard produced by Bio-Rad. The buffer solution used in the electrophoresis tank is Tris-glycine-SDS buffer solution, and the elution solution is 50mM NH4NO3And separating under the segmented voltage. The first period of voltage applied was 30 minutes 60V and the second period of voltage applied was 5 hours 200V, and the separation results are shown in FIGS. 4A and 4B, where FIG. 4A is the Precision Plus Protein Dual Color Protein produced by Bio-Rad according to the present inventionThe result of the standard substance separation; FIG. 4B is a diagram showing the effect of SDS-PAGE separation by Bio-Rad. As can be seen from the figure, the band formed by separating the protein standard substance by using the column-shaped gel electrophoresis device of the invention is very consistent with the separation result of commercial SDS-PAGE, which proves that the invention has very good protein separation capability.
Example 2
Two applications I were performed using the horizontal column gel electrophoresis apparatus of the present invention127Labeled standard proteins were isolated on-line and tested in conjunction with ICP-MS.
The gel tube used was filled with polyacrylamide gel at 40. mu.l concentration of 4% concentrate gel and 180. mu.l concentration of 10% separation gel, I127Both of the labeled standard proteins were purchased from Sigma, USA, and the sample loading was 20. mu.L, and the electrophoresis voltage program was 100V 10min, 200V 20min, and 600V 2h at a protein concentration of 0.01. mu.g/. mu.L. The electrophoresis buffer salt solution used was: Tris-glycine-SDS solution. The protein eluent was 50mM NH4NO3And (3) solution.
The rotation speed of the external peristaltic pump is 140 mu L/min, and the used ICP-MS is 8800 type ICP-MS produced by Agilent. The incident power was 1500W, the collision airflow rate was 15.0L/min, the carrier gas flow rate was 0.80L/min, the auxiliary airflow rate was 0.80L/min, the mist chamber temperature was 2 ℃, the mass number (m/z) was 127I, and the integration time was 0.1 sec.
The results are shown in FIG. 5, in which peak 1 is RA protein and peak 2 is CA protein. As can be seen from the figure, the column-shaped gel electrophoresis device of the invention can rapidly and accurately carry out the two applications I127The marked standard protein is separated and detected on line, and has better peak pattern and smaller dead volume.
The GE-ICP-MS online coupling system is shown in figure 1, and modifies an electrophoresis apparatus gel tube and an elution apparatus, and changes an original glass gel tube with the inner diameter of 6.5mm into a quartz tube 5 with the inner diameter of 2.5mm, so that the sample loading amount of a sample is reduced, and the separation degree and the resolution ratio are improved; the nylon sleeve pipe that utilizes 3D to print changes bottom filter core 9 size into 8.2mm as eluent drainage device 8, reduces solution dead volume to improve the resolution. After being separated and eluted, the protein sample is respectively introduced into the ICP-MS13 and the component collector 14 through a PEEK liquid phase pipeline tee joint 11 at a flow rate of 140 mu L/min by a peristaltic pump 12. And the sample enters ICP-MS through the fog chamber to detect the metal elements and the nonmetal elements. The component collector collects samples for subsequent protein identification and other detection.
Example 3
As shown in FIGS. 6A and 6B, the horizontal column gel electrophoresis apparatus of the present invention was used for three kinds of applications I127Respectively carrying out on-line separation and detection by combining with ICP-MS (inductively coupled plasma-mass spectrometry) on the labeled standard proteins, and detecting the same standard proteins by using an optimized commercial column gel electrophoresis device under the same conditions, wherein I is simultaneously detected due to the fact that the proteins possibly contain metal elements127And Zn65And the results are compared.
The polyacrylamide gel filled in the gel tube was 50. mu.l of 4% strength concentrated gel and 200. mu.l of 10% strength separation gel, and I was used127The three labeled standard proteins were purchased from Sigma, USA, and the sample loading was 20 μ L, and the electrophoresis voltage program was 100V 10min, 200V 20min, and 600V 2h at a protein concentration of 0.01 μ g/. mu.L. The electrophoresis buffer salt solution used was: Tris-glycine-SDS solution. The protein eluent was 50mM NH4NO3And (3) solution.
The rotation speed of the external peristaltic pump is 140 mu L/min, and the used ICP-MS is 8800 type ICP-MS produced by Agilent. The incident power was 1500W, the collision airflow rate was 15.0L/min, the carrier gas flow rate was 0.80L/min, the auxiliary airflow rate was 0.80L/min, the mist chamber temperature was 2 ℃, the mass number (m/z) was 127I, and the integration time was 0.1 sec.
The detection results are shown in fig. 6A and 6B, where fig. 6A is the separation and detection results of the device for three proteins, fig. 6B is the separation and detection results of the optimized commercial electrophoresis device for three proteins, and in the figure, peak 1 is RA protein, peak 2 is CA protein, and peak 3 is BSA protein. As can be seen from the figure, the column-shaped gel electrophoresis device of the invention can rapidly and accurately process three types of I127Labeled standard protein is performed on-lineSeparation and detection, and better peak pattern and response values than the optimized commercial electrophoresis device, which shows that the electrophoresis device and the components thereof have better protein separation efficiency and analysis sensitivity.
Multiple tests prove that the inner diameter of the gel tube is reduced by the horizontal cylindrical gel electrophoresis device for protein separation, so that the sample loading amount of a sample can be reduced, repeated experiment results show that the smaller inner diameter can improve the protein separation degree and the electrophoresis resolution, the separation condition selection range is wide, but the too small inner diameter is not beneficial to the gel filling; the horizontal electrophoresis tank for 3D printing has simple structure and low cost, and is easier to operate than the existing commercial electrophoresis tank; the elution device for 3D printing has low cost and simple manufacturing process, and experimental results show that the dead volume of the elution device is greatly reduced, so that the detection efficiency and sensitivity of protein are obviously improved, and disposable use or recycling use can be realized. The column gel electrophoresis device and the column gel tube, the electrophoresis tank and the elution device contained in the column gel electrophoresis device are matched for use and are combined with instruments such as ICP-MS and the like, so that the rapid and accurate off-line separation and on-line separation and detection of the protein can be realized.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (13)
1. A horizontal column gel electrophoresis apparatus for protein separation, the horizontal column gel electrophoresis apparatus employing a horizontal configuration, comprising:
a cylindrical gel tube filled with gel for protein sample separation; the length of the columnar gel tube is 8-12 cm; and
an electrophoresis tank including a buffer solution tank on a positive side, a buffer solution tank on a negative side, a cooling water tank located in the middle, and an elution solution tank located on the positive side; the electrophoresis tank is internally provided with a position for placing the columnar gel tube, so that the gel tube can be completely immersed in the cooling water tank, the columnar gel electrophoresis device formed by the electrophoresis tank is a horizontal columnar gel electrophoresis device, the columnar gel tube is horizontally placed, the wall of the buffer solution tank on the positive side, which is adjacent to the elution solution tank, is provided with an elution tube jack, the elution solution tank is used for providing solution for the elution device, and the electrophoresis tank is connected with a sample inlet of the inductively coupled plasma mass spectrometer through the elution device; and
the elution device comprises a front part and a rear part which can be assembled; the front end of the front half part of the elution device is provided with a jack of a cylindrical gel tube with the length of 6-8mm and the diameter of 5.5-7.5 mm; the middle of the front half part is a round hole with the length of 1.3mm and the diameter of 4mm, and is used for placing a first SPE sieve plate, and two sides of the round hole are respectively connected through an elution pipe; the rear end of the front half part is provided with a port with the length of 4.1mm and the diameter of 11.2mm, and the port is connected with the rear half part; a round hole with the length of 1.6mm and the diameter of 9.2mm is formed in the front end of the rear half part of the elution device and used for placing a second SPE sieve plate, and a dialysis membrane is placed at the front end of the second SPE sieve plate; the rear end of the rear half part is provided with a connecting hole communicated with the buffer solution storage container.
2. The horizontal cylindrical gel electrophoresis device according to claim 1 wherein the cylindrical gel tube has an outer diameter of 4-6 mm; the inner diameter of the columnar gel tube is 2-2.2 mm; the length of the columnar gel tube is 8 cm.
3. The horizontal column gel electrophoresis apparatus of claim 1 wherein said electrophoresis tank is made of a high molecular polymer material.
4. The horizontal cylindrical gel electrophoresis device of claim 1 wherein the electrophoresis tank has a depth of 3.5-5cm, a width of 8-12cm and a total length of 18-22 cm.
5. The horizontal column gel electrophoresis apparatus of claim 4, wherein the length of the cooling water tank in the electrophoresis tank is 5.5-9 cm; the lengths of the buffer solution tank and the elution solution tank on the positive electrode side are 5-6 cm; the length of the buffer solution tank on the negative electrode side is 5 to 6 cm.
6. The horizontal column gel electrophoresis apparatus according to claim 1, wherein electrode holders of positive and negative electrodes are provided outside the same side of the buffer solution tank on the positive side and the buffer solution tank on the negative side, respectively, and are equipped with electrodes.
7. The horizontal cylindrical gel electrophoresis apparatus according to claim 6, wherein a movable snap-gauge is provided at the top end of the outside of one side of the cooling water tank for fixing the cylindrical gel tube and closing the cooling water tank; and two screwing screw holes are respectively formed in one end of the movable clamping plate and the other side of the cooling water tank, and the movable clamping plate is screwed by inserting screws during use.
8. The horizontal column gel electrophoresis device of claim 1 wherein the main body of the elution device is made of a high molecular weight polymer material.
9. The horizontal column-shaped gel electrophoresis device according to claim 8, wherein the main body portion of the elution device is made of synthetic resin;
the elution device body portion is manufactured by 3D printing techniques.
10. A horizontal cylindrical gel electrophoresis device according to claim 1 wherein in use a cylindrical gel tube is inserted into the front half of the elution device and contacts the first SPE sieve deck.
11. The horizontal column gel electrophoresis device of claim 10 wherein said elution unit is used by assembling the front and back sections together and wrapping the interface with a sealing film.
12. An elution device for protein separation as claimed in claim 1 wherein the elution device requires sufficient wetting of the first SPE frit, second SPE frit and dialysis membrane and removal of air to ensure efficacy prior to use.
13. The horizontal column gel electrophoresis apparatus according to claim 1 wherein the elution apparatus is used with elution tubes at both upper and lower ends, the upper end elution tube is inserted into the elution solution tank through an elution tube insertion hole on the electrophoresis tank, the lower end elution tube is connected to a cannula at the peristaltic pump side, the elution solution enters from the upper end elution tube, passes through the first SPE sieve plate, is guided out of the elution apparatus by the lower end elution tube, and is introduced into the inductively coupled plasma mass spectrometer under the control of the flow rate of the peristaltic pump.
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