CN114397349B - Protein electrophoresis separation method based on agarose stacking gel - Google Patents
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 68
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 68
- 229920000936 Agarose Polymers 0.000 title claims abstract description 67
- 238000001962 electrophoresis Methods 0.000 title claims abstract description 49
- 238000000926 separation method Methods 0.000 title claims abstract description 28
- 239000000499 gel Substances 0.000 claims abstract description 89
- 239000007853 buffer solution Substances 0.000 claims abstract description 25
- 239000011543 agarose gel Substances 0.000 claims abstract description 21
- 239000011521 glass Substances 0.000 claims description 51
- 239000003292 glue Substances 0.000 claims description 49
- 238000011049 filling Methods 0.000 claims description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000000741 silica gel Substances 0.000 claims description 10
- 229910002027 silica gel Inorganic materials 0.000 claims description 10
- 238000001502 gel electrophoresis Methods 0.000 claims description 9
- 238000005266 casting Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 8
- 238000010438 heat treatment Methods 0.000 claims description 6
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 4
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 4
- 239000012192 staining solution Substances 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 3
- 229920002401 polyacrylamide Polymers 0.000 description 10
- 238000000605 extraction Methods 0.000 description 8
- 239000012474 protein marker Substances 0.000 description 7
- 108010074124 Escherichia coli Proteins Proteins 0.000 description 6
- 238000005485 electric heating Methods 0.000 description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 4
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 4
- 102100027340 Slit homolog 2 protein Human genes 0.000 description 3
- 101710133576 Slit homolog 2 protein Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000007692 polyacrylamide-agarose gel electrophoresis Methods 0.000 description 2
- 238000007711 solidification Methods 0.000 description 2
- 230000008023 solidification Effects 0.000 description 2
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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Abstract
The invention belongs to the technical field of biology, and relates to a protein electrophoresis separation method based on agarose stacking gel. The method has simple process and simple and convenient operation, and agarose is matched with corresponding gel buffer solution, high-concentration agarose gel is vertically stacked in a stacking mode, and the agarose gel can be used for carrying out electrophoresis separation on protein under the condition of higher concentration by utilizing the set electrophoresis conditions, so that the application range of agarose gel electrophoresis in protein separation is improved.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a protein separation method, in particular to a method for carrying out electrophoresis separation on proteins by adopting high-concentration agarose stacking gel.
Background
Proteins exist in all living bodies as important substances for vital activities, play a vital role in the life cycle of the living bodies, and play a role in various physiological functions such as biocatalysis, substance transportation, exercise, defense, regulation, memory, recognition and the like, so that research techniques for proteins are a key point in the field of life science. Because the structure and function of the protein are studied deeply, the separation and purification of the protein are core technologies in the biological industry, and the purified protein sample is obtained by separating the protein, so that the identification and study on the structure, the physical and chemical properties and the like of the protein can be performed.
Electrophoresis is a common method of separating biological macromolecular substances, and the electrophoresis support medium commonly used in the process of electrophoresis separation is a gel, such as agarose gel, polyacrylamide gel. In general, the gel used for separating nucleic acid molecules by electrophoresis is agarose gel, and the gel used for separating protein molecules by electrophoresis is polyacrylamide gel. The separation degree of the protein and the nucleic acid is approximately the same for the polyacrylamide gel and the agarose gel with the same concentration, but the polyacrylamide gel is more suitable for preparing the gel with higher concentration (more than or equal to 4 percent), and the agarose gel is more suitable for preparing the gel with lower concentration (less than or equal to 4 percent), so the polyacrylamide gel is used for separating the protein with smaller molecular weight, and the agarose gel is used for separating the nucleic acid molecules with larger molecular weight. Moreover, in addition to the difficulty in uniform melting of high concentration agarose in the preparation of agarose vertical gels, there are three bottleneck problems: one is how easy the viscous agarose sol is poured into the ultra-narrow gap of the vertical glass plate; secondly, when the lower layer gel of the agarose stacked gel is prepared, the gel surface cannot be horizontal, and the horizontal state of the overlapped surface of the upper layer gel and the lower layer gel is ensured; thirdly, selecting what buffer solution is used for high-concentration agarose stacking gel electrophoresis under what electrophoresis condition. For these reasons, only medium-low concentration agarose gel has been used for separating large molecular weight proteins or protein complexes, and the application range of agarose gel electrophoresis in protein separation is greatly limited.
Disclosure of Invention
The invention aims to provide a protein electrophoresis separation method based on agarose stacking gel, which aims at the defects of the prior art, prepares agarose stacking vertical gel by agarose matching with corresponding buffer solution in a stacking way, can carry out electrophoresis separation on protein by using agarose gel under the condition of higher concentration by using set electrophoresis conditions, and improves the application range of agarose gel electrophoresis in protein separation.
The invention adopts the technical scheme that:
a protein electrophoresis separation method based on agarose stacking gel comprises the following steps:
1. preparation of gel before casting
Preparing gel buffer solution with Tris-Cl and NaCl at the concentration of 0.25M Tris-Cl and 0.04-0.2% NaCl for standby;
preparing electrophoresis buffer solution with Tris-Cl and SDS at the concentration of 0.25M Tris-Cl and 0.2-1.0% SDS for standby;
and (3) manufacturing a top layer glue box: taking a rectangular box, wherein the length of the rectangular box is slightly smaller than the length of an inner hole of the glass plate glue-pouring device, a thin seam is formed in the bottom of the box and parallel to the long side direction, the width of the thin seam is slightly larger than the width of the inner hole of the glass plate glue-pouring device, two silica gel strips are symmetrically arranged on the two sides of the thin seam at the outer side of the bottom of the box, and the distance between the silica gel strips is equal to the thickness of the glass plate glue-pouring device.
2. Agarose stacking gel casting:
s1, pouring agarose stacked gel lower gel: according to the preparation standard of agarose solution with concentration of 8-14%, mixing gel buffer solution and agarose, and dissolving in steam environment under high temperature and high pressure to obtain clarified 8-14% agarose sol for standby; preheating a glass plate glue filling device and a top layer glue box at 75-85 ℃, then vertically placing the glass plate glue filling device in an environment of 75-85 ℃, installing the top layer glue box at the top of the glass plate glue filling device, aligning a thin seam at the bottom of the top layer glue box with an inner hole (a flat hole between glass plates) of the glass plate glue filling device, pouring agarose sol with the temperature of 75-85 ℃ into the top layer glue box, and standing for 20-40 min at room temperature after the sol flows into the inner hole of the glass plate glue filling device, and waiting for the sol to solidify.
In the agarose sol preparation process, the high temperature and high pressure refer to the temperature of 115-121 ℃ and the pressure of 1.6X10 5 Pa～2.1×10 5 Pa。
S2, pouring agarose stacking gel upper gel:
after the sol is solidified, pushing the gel out of the glass plate glue-pouring device from bottom to top by using a comb for 2.5-3.0 cm, and then aligning the edge of the glass plate glue-pouring device to cut off the pushed agarose gel; inverting the glass plate glue filling device by 180 degrees, taking out the comb, and preheating again at 75-85 ℃ for 3-5 minutes; according to the preparation standard of agarose solution with the concentration of 3-4%, mixing gel buffer solution and agarose, heating and dissolving to obtain 3-4% agarose sol, injecting 3-4% agarose sol into the inner hole of a glass plate glue-pouring device from the upper part, inserting a comb to 1.5-2 cm, standing at room temperature for 20-40 min, and waiting for complete solidification of the sol.
3. Agarose stacking gel electrophoresis separation of proteins:
and (3) after a target protein sample is sampled in an electrophoresis apparatus, carrying out constant voltage electrophoresis for 4-5 hours by using an electrophoresis buffer solution according to the standard of the gel length of 9-11V/cm until the front edge of bromophenol blue reaches the bottom of the gel, putting the gel into coomassie brilliant blue R-250 staining solution for dyeing overnight, transferring the gel into decolorizing solution for decolorizing for 4-5 hours, and scanning by using a gel scanner to obtain electrophoresis patterns, thus obtaining different protein strips.
The method has simple process and simple and convenient operation, and agarose is matched with corresponding gel buffer solution, high-concentration agarose gel is vertically stacked in a stacking mode, and the agarose gel can be used for carrying out electrophoresis separation on protein under the condition of higher concentration by utilizing the set electrophoresis conditions, so that the application range of agarose gel electrophoresis in protein separation is improved.
Drawings
Fig. 1 is a schematic top view of the top-level cartridge of the present invention.
In the figure: 1. a rectangular box; 2. a slit; 3. and (5) a silica gel strip.
Fig. 2 is a schematic side view of the top layer cartridge of the present invention.
In the figure: 1. a rectangular box; 3. and (5) a silica gel strip.
FIG. 3 is a comparison of the separation effect of 8% polyacrylamide gel electrophoresis and agarose gel electrophoresis on proteins. PAGE, polyacrylamide stacking gel electrophoresis; agarose, agarose stack gel electrophoresis. a. 10-170kDa protein molecular weight standard; b. 6.5-270kDa protein molecular weight standard; c. e.coli proteins; d. and adding the IPTG induced escherichia coli protein.
FIG. 4 is a comparison of the separation effect of 12% polyacrylamide gel electrophoresis and agarose gel electrophoresis on proteins. PAGE: polyacrylamide stacking gel electrophoresis; boost: agarose stacking gel electrophoresis. A. Protein markers with 10-170kDa protein molecular weight standard; B. 6.5-270kDa protein molecular weight standard protein Marker; C. e.coli proteins; D. and adding the IPTG induced escherichia coli protein.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer.
And (3) manufacturing a top layer glue box: as shown in fig. 1 and 2, a PP plastic rectangular box 1 with a length slightly smaller than the inner hole length of the glass plate glue-pouring device is purchased (the inner hole specifications of the glass plate glue-pouring devices produced by different manufacturers are different), a slit 2 is formed in the bottom of the box in parallel to the long side direction, the width of the slit 2 is slightly larger than the inner hole width of the glass plate glue-pouring device, two silica gel strips 3 are symmetrically arranged on two sides of the slit 2 at the outer side of the bottom of the box, and the distance between the silica gel strips is equal to the thickness of the glass plate glue-pouring device.
Example 1
1. Preparation of gel before casting
Preparing gel buffer solution with Tris-Cl and NaCl at concentration of 0.25M Tris-Cl and 0.1% NaCl respectively for use;
preparing electrophoresis buffer solution with Tris-Cl and SDS at concentration of 0.25M Tris-Cl and 0.5% SDS respectively for use;
2. agarose stacking gel casting:
s1, pouring agarose stacked gel lower gel: according to the preparation standard of agarose solution with concentration of 8%, placing gel buffer solution and agarose into reagent bottle, mixing, placing into autoclave, setting temperature at 115 deg.C (correspondent pressure is 1.68X10) 5 Pa), heating for 20min to dissolve agarose to obtain clear 8% agarose sol for later use; placing the glass plate glue injector and the top layer glue box into an electric heating box, preheating at 80 ℃, then vertically placing the glass plate glue injector in the electric heating box (controlling the temperature to be about 80 ℃), installing the top layer glue box on the top of the glass plate glue injector (using two silica gel strips at the bottom of the top layer glue box to clamp the outer side of the glass plate glue injector), aligning a thin seam at the bottom of the top layer glue box with an inner hole of the glass plate glue injector, and cooling to 8 DEG CPouring the 8% agarose sol at about 0 ℃ into a top layer glue box, standing for 30min at room temperature after the sol flows into the inner hole of the glass plate glue filling device, and waiting for the sol to solidify.
S2, pouring agarose stacking gel upper gel:
after the sol is solidified, pushing the gel out of the glass plate glue-pouring device from bottom to top by using a comb for 2.5-3.0 cm, and then aligning the edge of the glass plate glue-pouring device to cut off the pushed agarose gel; inverting the glass plate glue filling device by 180 degrees, taking out the comb, putting the comb into an electric heating box again, and preheating for 4 minutes at 75-85 ℃; according to the preparation standard of agarose gel with the concentration of 4%, mixing a gel buffer solution and agarose, heating and dissolving to obtain 4% agarose gel, injecting the 4% agarose gel into an inner hole of a glass plate glue filling device from the upper part, inserting a comb to 1.5-2 cm, standing at room temperature for 40min, and waiting for the sol to be completely solidified.
3. Agarose stacking gel electrophoresis separation of proteins:
the four proteins are respectively taken from a protein Marker (commercially available, marked as a) with a molecular weight standard of 10-170kDa protein, a protein Marker (commercially available, marked as b) with a molecular weight standard of 6.5-270kDa protein, escherichia coli protein (self-extraction, marked as c) and escherichia coli protein (self-extraction, marked as d) induced by adding IPTG.
After loading a target protein sample in an electrophoresis apparatus, carrying out constant voltage electrophoresis on the target protein sample for 4 hours by using an electrophoresis buffer solution according to the standard of the gel length of 10V/cm until the front edge of bromophenol blue reaches the bottom of the gel, putting the gel into a coomassie brilliant blue R-250 staining solution for dyeing overnight, transferring the gel into a decolorizing solution for decolorizing for 5 hours, and scanning by using a gel scanner [ particularly EXPRESSION 10000XL (Epson) ] to obtain an electrophoresis pattern, as shown in figure 3.
Example two
1. Preparation of gel before casting
Preparing gel buffer solution with Tris-Cl and NaCl at the concentration of 0.25M Tris-Cl and 0.2% NaCl respectively for later use;
preparing electrophoresis buffer solution with Tris-Cl and SDS at concentration of 0.25M Tris-Cl and 1.0% SDS respectively for use;
2. agarose stacking gel casting:
s1, pouring agarose stacked gel lower gel: according to the preparation standard of agarose solution with concentration of 12%, placing gel buffer solution and agarose into reagent bottle, mixing, placing into autoclave, setting temperature to 121 deg.C (correspondent pressure is 2.05X10) 5 Pa), heating for 20min to dissolve agarose to obtain clear 12% agarose sol for later use; placing the glass plate glue filling device and the top layer glue box into an electric heating box, preheating at 85 ℃, then vertically placing the glass plate glue filling device in the electric heating box (controlling the temperature to be about 80 ℃), installing the top layer glue box at the top of the glass plate glue filling device (using two silica gel strips at the bottom of the top layer glue box to clamp the outer sides of the glue filling glass plate), aligning a thin seam at the bottom of the top layer glue box with an inner hole of the glass plate glue filling device, pouring 12% agarose sol cooled to about 80 ℃ into the top layer glue box, standing at room temperature for 30min after the sol flows into the inner hole of the glass plate glue filling device, and waiting for the sol to solidify.
S2, pouring agarose stacking gel upper gel:
after the sol is solidified, pushing the gel out of the glass plate glue-pouring device from bottom to top by using a comb for 2.5-3.0 cm, and then aligning the edge of the glass plate glue-pouring device to cut off the pushed agarose gel; inverting the glass plate glue filling device by 180 degrees, taking out the comb, putting the comb into an electric heating box again, and preheating for 5 minutes at 80 ℃; according to the preparation standard of agarose solution with the concentration of 4%, mixing gel buffer solution and agarose, heating and dissolving to obtain 4% agarose sol, injecting the 4% agarose sol into the inner hole of a glass plate glue-pouring device from the upper part, inserting a comb to 1.5-2 cm, standing at room temperature for 40min, and waiting for complete solidification of the sol.
3. Agarose stacking gel electrophoresis separation of proteins:
the four proteins are respectively taken from a protein Marker (commercially available and marked as A) with a protein molecular weight standard of 10-170kDa, a protein Marker (commercially available and marked as B) with a protein molecular weight standard of 6.5-270kDa, escherichia coli protein (self-extraction and marked as C) and escherichia coli protein (self-extraction and marked as D) after being added with IPTG induction.
After loading a target protein sample in an electrophoresis apparatus, carrying out constant voltage electrophoresis for 4 hours by using an electrophoresis buffer according to the standard of the gel length of 11V/cm until the front edge of bromophenol blue reaches the bottom of the gel, putting the gel into a coomassie brilliant blue R-250 staining solution for dyeing overnight, transferring the gel into a decolorizing solution for decolorizing for 5 hours, and scanning by using a gel scanner [ particularly EXPRESSION 10000XL of Epson company ], thereby obtaining an electrophoresis pattern, as shown in figure 4.
Comparison test:
comparative example one
The electrophoresis pattern is obtained by using high concentration polyacrylamide gel (8%) as electrophoresis support medium, using protein Marker (commercially available, marked as a) with 10-170kDa protein molecular weight standard, protein Marker (commercially available, marked as b) with 6.5-270kDa protein molecular weight standard, coliform protein (self-extraction, marked as c), and coliform protein induced by adding IPTG (self-extraction, marked as d), and four kinds of proteins as target proteins (the sources of target proteins are the same as in the first embodiment), and adopting conventional electrophoresis separation process, as shown in figure 3.
Comparative example two:
the electrophoresis pattern is obtained by using high-concentration polyacrylamide gel (12%) as electrophoresis support medium, using protein markers (commercially available, marked as A) with 10-170kDa protein molecular weight standard, protein markers (commercially available, marked as B) with 6.5-270kDa protein molecular weight standard, coliform protein (self-extraction, marked as C), and coliform protein induced by adding IPTG (self-extraction, marked as D), and using conventional electrophoresis separation process to obtain electrophoresis pattern, as shown in figure 4.
The result of the protein electrophoresis pattern shows that the high-concentration agarose gel is vertically stacked by adopting a proper gel buffer solution in a stacking mode, the protein can be separated by electrophoresis by using the agarose gel under the condition of higher concentration, the separation effect is similar to that of the high-concentration polyacrylamide gel, the high-concentration agarose gel has higher band resolution, and the application range of agarose gel electrophoresis in protein separation is improved.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (1)
1. The protein electrophoresis separation method based on agarose stacking gel is characterized by comprising the following steps:
1) Preparation before gel casting
Preparing gel buffer solution with Tris-Cl and NaCl at the concentration of 0.25M Tris-Cl and 0.04-0.2% NaCl for standby;
preparing electrophoresis buffer solution with Tris-Cl and SDS, wherein the concentration of the electrophoresis buffer solution is 0.25M Tris-Cl and 0.2-1.0% SDS respectively for standby;
and (3) manufacturing a top layer glue box: taking a rectangular box, wherein the length of the rectangular box is slightly smaller than the length of an inner hole of the glass plate glue-pouring device, a thin seam is formed in the bottom of the box and parallel to the long side direction, the width of the thin seam is slightly larger than the width of the inner hole of the glass plate glue-pouring device, two silica gel strips are symmetrically arranged on the two sides of the thin seam at the outer side of the bottom of the box, and the distance between the silica gel strips is equal to the thickness of the glass plate glue-pouring device;
2) Agarose stack gel casting:
s1, pouring agarose stacked gel lower gel: according to the preparation standard of agarose solution with concentration of 8-14%, mixing gel buffer solution and agarose, and dissolving in steam environment under high temperature and high pressure to obtain clarified 8-14% agarose sol for standby; preheating a glass plate glue filling device and a top layer glue box at 75-85 ℃, then vertically placing the glass plate glue filling device in an environment of 75-85 ℃, installing the top layer glue box at the top of the glass plate glue filling device, aligning a thin seam at the bottom of the top layer glue box with an inner hole of the glass plate glue filling device, pouring agarose sol with the temperature of 75-85 ℃ into the top layer glue box, placing the glass plate glue filling device at room temperature for 20-40 min after the sol flows into the inner hole of the glass plate glue filling device, and waiting for the sol to solidify; the high temperature and high pressure refer to the temperature of 115-121 ℃ and the pressure of 1.6X10 5 Pa～2.1×10 5 Pa；
S2, pouring agarose stacking gel upper gel:
after the sol is solidified, pushing the gel out of the glass plate glue-pouring device from bottom to top by using a comb for 2.5-3.0 cm, and then aligning the edge of the glass plate glue-pouring device to cut off the pushed agarose gel; inverting the glass plate glue filling device by 180 degrees, taking out the comb, and preheating again at 75-85 ℃ for 3-5 minutes; according to the preparation standard of agarose solution with the concentration of 3-4%, mixing gel buffer solution and agarose, heating and dissolving to obtain 3-4% agarose sol, injecting 3-4% agarose sol into the inner hole of a glass plate glue-pouring device from the upper part, inserting a comb to 1.5-2 cm, standing at room temperature for 20-40 min, and waiting for the sol to be completely solidified;
3) Agarose stacked gel electrophoresis separation of proteins:
and (3) after a target protein sample is sampled in an electrophoresis apparatus, carrying out constant voltage electrophoresis for 4-5 hours by using an electrophoresis buffer solution according to the standard of the gel length of 9-11V/cm until the front edge of bromophenol blue reaches the bottom of the gel, putting the gel into coomassie brilliant blue R-250 staining solution for dyeing overnight, transferring the gel into decolorizing solution for decolorizing for 4-5 hours, and scanning by using a gel scanner to obtain electrophoresis patterns, thus obtaining different protein strips.
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| US5009759A (en) * | 1989-09-22 | 1991-04-23 | Board Of Regents, The University Of Texas System | Methods for producing agarose gels having variable pore sizes |
| CN101788541A (en) * | 2009-01-24 | 2010-07-28 | 复旦大学 | Method for separating, enriching and identifying macromolecular weight protein |
| CN101701933A (en) * | 2009-10-19 | 2010-05-05 | 王永杰 | non-concentrated urine protein electrophoresis agarose gel plate |
| CN103952302A (en) * | 2014-04-15 | 2014-07-30 | 同济大学 | Novel substrate for single-cell gel eletrophoresis experiments |
| CN108251550A (en) * | 2017-12-12 | 2018-07-06 | 贵州省烟草科学研究院 | A kind of method of HRM detection tobacco cadmium transporter genes NtHMA4 mutation |
| CN209979547U (en) * | 2018-12-25 | 2020-01-21 | 上海循益生物技术有限公司 | Horizontal agarose electrophoresis device for DNA detection and gel maker thereof |
| CN110935403A (en) * | 2019-12-07 | 2020-03-31 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently preparing agarose gel |
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