Gel electrophoresis kit and preparation method thereof

Abstract

The invention provides a gel electrophoresis kit which comprises the following components: concentrated gel which comprises 30% acrylamide / methylene bisacrylamide (29: 1) with the concentration of 5-30%; the photoinitiator is used for solidifying the liquid concentrated gel under the irradiation of a light source of 365 nm to 405 nm; the separation gel comprises the following components: 30% acrylamide / methylene bisacrylamide (29: 1) with the concentration of 10%-65%; the photoinitiator is used for solidifying the liquid separation gel under the irradiation of a light source of 365 nm to 405 nm; a concentrated gel buffer solution; and separating a gel buffer solution. The invention also provides a preparation method of the gel electrophoresis kit. According to the gel electrophoresis kit provided by the invention, the photoinitiator used when the separation gel and the concentration gel are prepared can be excited to generate photochemical change under the irradiation of a light source so as to generate an active intermediate with the capability of initiating polymerization, and the active intermediate can promote the solidification of liquid gel. The gel solidification speed under the excitation of the photoinitiator is obviously higher than that of natural solidification, and the experiment speed is accelerated.

Description

Technical Field

[0001] The present application relates to the technical field of protein electrophoresis reagents, and in particular to a gel electrophoresis kit and a preparation method thereof. Background Art

[0002] Protein electrophoresis is one of the core tools in biological research and is widely used in the fields of protein separation, molecular weight determination and purity identification. Traditional protein electrophoresis often uses polyacrylamide gel to achieve protein separation. This type of gel is generally composed of two parts: a concentration gel and a separation gel. The former plays a role in concentrating protein samples, while the latter separates proteins of different sizes based on the different concentrations of acrylamide monomers and N,N-methylene acrylamide (methyl acrylamide) crosslinkers used in the gel.

[0003] In the process of preparing the stacking gel and separation gel, 10% APS (ammonium persulfate) and TEMED (tetramethyl diethylamine) need to be added to polymerize acrylamide and N, N-methylene acrylamide respectively. In the process of polymerization of the stacking gel and separation gel, a lot of waiting time is required. This time-consuming gel preparation limits its application efficiency in high-throughput experiments. Summary of the invention

[0004] The present application provides a gel electrophoresis kit and a preparation method thereof, so as to solve the problem that a lot of waiting time is required in the process of preparing the gel electrophoresis kit during the polymerization of the concentrated gel and the separated gel.

[0005] In order to solve the above technical problems, the present application provides a gel electrophoresis kit, comprising: a concentrated gel, whose components include: 30% acrylamide / methylene bisacrylamide (29:1) with a concentration of 5%-30%; a photoinitiator, wherein the photoinitiator solidifies the liquid concentrated gel under the irradiation of a light source of 365nm-405nm; a separation gel, whose components include: acrylamide / methylene bisacrylamide (29:1) with a concentration of 10%-65%; a photoinitiator, wherein the photoinitiator solidifies the liquid separation gel under the irradiation of a light source of 365nm-405nm; a concentrated gel buffer; and a separation gel buffer.

[0006] Furthermore, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, and its concentration is 0.0001%-1%, wherein the optimal concentration is 0.005%.

[0007] Furthermore, the concentrated gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-2%, the pH value of the concentrated gel buffer is 6.7-6.9, and the concentration of the concentrated gel buffer is 5%-30%.

[0008] Furthermore, the separation gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-5%, the pH value of the separation gel buffer is 8.7-8.9, and the concentration of the separation gel buffer is 10%-40%.

[0009] The present invention also provides a method for preparing a gel electrophoresis kit, comprising:

[0010] Mixing acrylamide / methylenebisacrylamide (29:1) with a component concentration of 10% to 65%, a photoinitiator and a separation gel buffer to form a separation gel;

[0011] Adding a concentrated gel formed by mixing 30% acrylamide / methylenebisacrylamide (29:1) with a component concentration of 5%-30%, a photoinitiator and a concentrated gel buffer onto the separation gel to form a layered solution;

[0012] The layered solution is irradiated with a light source of 365 nm to 405 nm to solidify the layered solution.

[0013] Furthermore, in the preparation method of the gel electrophoresis kit, the photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, and its concentration is 0.0001%-1%, wherein the optimal concentration is 0.005%.

[0014] Furthermore, in the preparation method of the gel electrophoresis kit, the stacking gel buffer comprises: tris(hydroxymethylaminomethane), hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-2%, the pH value of the stacking gel buffer is 6.7-6.9, and the concentration of the stacking gel buffer is 5%-30%.

[0015] Furthermore, in the preparation method of the gel electrophoresis kit, the separation gel buffer comprises: tris(hydroxymethylaminomethane), hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-5%, the pH value of the separation gel buffer is 8.7-8.9, and the concentration of the separation gel buffer is 10%-40%.

[0016] Furthermore, in the preparation method of the gel electrophoresis kit, the distance between the gel solution and the light source is 2 to 4 cm.

[0017] The photoinitiator used in the gel electrophoresis kit for preparing the separation gel and the concentrated gel can be excited to undergo photochemical changes under the irradiation of a light source to produce an active intermediate with the ability to initiate polymerization, and the active intermediate can promote the solidification of the liquid gel. The gel solidification speed under the excitation of the photoinitiator is significantly faster than the natural solidification, which speeds up the experiment.

[0018] The preparation method of the gel electrophoresis kit provided by the present invention can prepare the separation gel and the concentrated gel separately in advance. When the separation gel and the concentrated gel are needed to configure the gel kit for protein electrophoresis, the concentrated gel is added to the separation gel to form a layered solution, and the layered solution can be directly used after being irradiated for a short time. The preparation time is short, the experimental speed can be accelerated, and there is no need to wait for a long coagulation time when the catalyst is used to configure the gel as in the traditional process. DETAILED DESCRIPTION

[0019] The technical solutions in this application are described clearly and completely below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] Unless otherwise specified, the test methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] The present application provides a gel electrophoresis kit, comprising: a concentrated gel, the components of which include: acrylamide / methylene bisacrylamide (29:1) with a concentration of 5%-30%; a photoinitiator, wherein the photoinitiator solidifies the liquid concentrated gel under irradiation with a light source of 365nm-405nm; a separation gel, the components of which include: acrylamide / methylene bisacrylamide (29:1) with a concentration of 10%-65%; a photoinitiator, wherein the photoinitiator solidifies the liquid separation gel under irradiation with a light source of 365nm-405nm; a concentrated gel buffer; and a separation gel buffer.

[0023] In an embodiment of the present invention, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (2% LAP), and its concentration is 0.0001%-1%, wherein the optimal concentration is 0.005%. The concentrated gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, and the pH value of the concentrated gel buffer is 6.7-6.9. The separation gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, and the pH value of the separation gel buffer is 8.7-8.9.

[0024] In the embodiment of the present invention, the concentration of the separation gel is 4%-20%, wherein the required concentration percentages of the components are: distilled water is 10%-65%, acrylamide / methylene bisacrylamide (29:1) is 10%-65%, Tris-HCL-SDS separation gel buffer at pH 8.8 is 10%-40%, the concentration of 10% SDS (sodium dodecyl sulfate) is 0.1-5%, and the concentration of 2% LAP is 0.0001%-1%; the concentration of the stacking gel is 17%, wherein the required concentration percentages of the components are: distilled water is 40%-80%, 30% acrylamide / methylene bisacrylamide (29:1) is 5%-30%, Tris-HCL-SDS stacking gel buffer at pH 6.8 is 5%-30%, the concentration of 10% SDS (sodium dodecyl sulfate) is 0.1-2%, and the concentration of 2% LAP is 0.0001%-1%.

[0025] Table 1 shows the volumes of the components required to prepare separation gels of different concentrations (8% to 15%).

[0026]

[0027]

[0028] Table 2 shows the volumes of the components required to prepare the concentrated gel.

[0029] Table 2

[0030]

[0031] According to the separation gel and concentrated gel configured as above, when protein electrophoresis is required, the separation gel and concentrated gel of the required concentration are mixed, and under the irradiation of 365nm, 395nm, and 405nm light sources, the photoinitiator contained in the separation gel and concentrated gel is excited to undergo photochemical changes, generating active intermediates with the ability to initiate polymerization, and promoting the solidification of the mixed gel liquid. The gel electrophoresis kit provided in the embodiment of the present invention can be used in different gel systems, such as Bis-Tris, Tris-glycine, and Tris-acetic acid.

[0032] The present invention also provides a method for preparing a gel electrophoresis kit, comprising:

[0033] Mixing acrylamide / methylenebisacrylamide (29:1) with a component concentration of 10% to 65%, a photoinitiator and a separation gel buffer to form a separation gel;

[0034] Adding a concentrated gel formed by mixing 30% acrylamide / methylenebisacrylamide (29:1) with a component concentration of 5%-30%, a photoinitiator and a concentrated gel buffer onto the separation gel to form a layered solution;

[0035] The layered solution is irradiated with a light source of 365 nm to 405 nm to solidify the layered solution.

[0036] In an embodiment of the present invention, the separation gel is added to a gel-making glass plate, and then the concentrated gel is added, a comb is vertically inserted into the concentrated gel, the overflowed liquid is wiped off with paper, the gel-making glass plate is fixed on a gel-making rack, and the gel-making rack is placed in a gel system, that is, the gel-making rack is placed under irradiation with a 365nm-405nm light source, the distance between the gel-making rack and the light source is 2 to 4 cm, and the gel-making is completed after 3 minutes of illumination.

[0037] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection created by this application.

Claims

1. A gel electrophoresis kit, characterized in that: include: Concentrated gel, the components include: 30% acrylamide / methylenebisacrylamide (29:1) at a concentration of 5%-30%; A photoinitiator, wherein the photoinitiator solidifies the liquid concentrated gel under irradiation with a light source of 365nm-405nm; Separation gel, components include: 30% acrylamide / methylenebisacrylamide (29:1) at a concentration of 10%-65%; A photoinitiator, wherein the photoinitiator solidifies the liquid separation gel under irradiation with a light source of 365nm-405nm; Stacking gel buffer; Separating gel buffer.

2. The gel electrophoresis kit according to claim 1, characterized in that The photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, and its concentration is 0.0001%-1%, wherein the optimal concentration is 0.005%.

3. The gel electrophoresis kit according to claim 1, characterized in that The concentrated gel buffer comprises tris(hydroxymethyl)aminomethane, hydrochloric acid and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-2%, the pH value of the concentrated gel buffer is 6.7-6.9, and the concentration of the concentrated gel buffer is 5%-30%.

4. The gel electrophoresis kit according to claim 1, characterized in that The separation gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-5%, the pH value of the separation gel buffer is 8.7-8.9, and the concentration of the separation gel buffer is 10%-40%.

5. A method for preparing a gel electrophoresis kit, characterized in that: include: Mixing acrylamide / methylenebisacrylamide (29:1) with a component concentration of 10% to 65%, a photoinitiator and a separation gel buffer to form a separation gel; Adding a concentrated gel formed by mixing 30% acrylamide / methylenebisacrylamide (29:1) with a component concentration of 5%-30%, a photoinitiator and a concentrated gel buffer onto the separation gel to form a layered solution; The layered solution is irradiated with a light source of 365 nm to 405 nm to solidify the layered solution.

6. The method for preparing a gel electrophoresis kit according to claim 5, characterized in that: The photoinitiator is phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite, and its concentration is 0.0001%-1%, wherein the optimal concentration is 0.005%.

7. The method for preparing a gel electrophoresis kit according to claim 5, characterized in that: The concentrated gel buffer comprises tris(hydroxymethyl)aminomethane, hydrochloric acid and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-2%, the pH value of the concentrated gel buffer is 6.7-6.9, and the concentration of the concentrated gel buffer is 5%-30%.

8. The method for preparing a gel electrophoresis kit according to claim 5, characterized in that: The separation gel buffer comprises: tris(hydroxymethyl)aminomethane, hydrochloric acid, and sodium dodecyl sulfate, wherein the concentration of the sodium dodecyl sulfate is 0.1%-5%, the pH value of the separation gel buffer is 8.7-8.9, and the concentration of the separation gel buffer is 10%-40%.

9. The method for preparing a gel electrophoresis kit according to claim 5, characterized in that: The distance between the gel solution and the light source is 2 to 4 cm.

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