CN112444552A - Color-changeable polyacrylamide gel and kit containing same - Google Patents
Color-changeable polyacrylamide gel and kit containing same Download PDFInfo
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Abstract
The invention discloses a color-changeable polyacrylamide gel and a kit containing the same, wherein the color-changeable polyacrylamide gel comprises separation gel and concentrated gel, wherein the separation gel comprises 6-15% of polyacrylamide, 0.375M Tris-HCl, 0.1% SDS, 10% of ammonium persulfate, 0.1% of tetraethylethylenediamine and 0.002-0.01% of colorant; the concentrated gel comprises 5% of polyacrylamide, 0.25M Tris-HCl, 0.1% of SDS, 0.1% of ammonium persulfate, 0.1% of tetraethylethylenediamine and 0.02% -0.1% of colorant. The design of reducing the toxicity of the accelerator in the preparation process of the color-changeable polyacrylamide gel ensures that experimenters are not damaged by volatile toxic compounds; meanwhile, the coloring agents are respectively added into the separation gel and the concentrated gel, so that a user can conveniently judge whether the concentrated gel and the separation gel are solidified and loaded, the protein separation effect is not influenced, the gel preparation reagents are mixed in advance, the gel preparation process is accelerated, and the experiment efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of electrophoresis, and particularly relates to a color-changeable polyacrylamide gel kit.
Background
Polyacrylamide gel electrophoresis (PAGE) is the most extensive and fundamental experiment among the current protein experiments, and includes Native-PAGE and SDS-PAGE. According to the traditional PAGE, acrylamide and methylene acrylamide are polymerized by taking Tetramethylethylenediamine (TEMED) as an accelerator under the catalytic action of Ammonium Persulfate (APS), in the polymerization process, TEMED catalyzes ammonium persulfate to generate free radicals which initiate polymerization of acrylamide monomers, and methylene bond crosslinking is generated between methylene bisacrylamide and acrylamide chains to form a three-dimensional network structure, so that the acrylamide gel is prepared.
The traditional acrylamide gel has the following defects that TEMED is volatile, inflammable, corrosive, and strong in neurotoxicity and pungent smell; the concentrated gel is colorless, so that a sample is easily added out of a sample hole during sample loading, thereby causing pollution among samples and influencing the identification result after protein separation; whether the separation glue and the concentrated glue are solidified cannot be judged, the time is generally waited for 30min to 1h, and a large amount of time is wasted; if the waiting time is reduced, the gel preparation is easy to fail because the separation gel and the concentrated gel are not completely solidified; the glue making process is complex, various solutions need to be mixed, the glue making process is time-consuming and labor-consuming, and the experiment efficiency is reduced. In summary, a safer and more efficient rapid polyacrylamide gel preparation kit is urgently needed for the traditional polyacrylamide gel.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides a color-changeable polyacrylamide gel and a kit containing the same.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a colored polyacrylamide gel comprising a separation gel and a concentration gel, wherein the separation gel comprises 6-15% polyacrylamide, 0.375M Tris-HCl, 0.1% SDS, 10% ammonium persulfate, 0.1% tetraethylethylenediamine, 0.002% to 0.01% colorant; the concentrated gel comprises 5% of polyacrylamide, 0.25M Tris-HCl, 0.1% of SDS, 0.1% of ammonium persulfate, 0.1% of tetraethylethylenediamine and 0.02-0.1% of colorant.
Specifically, the polyacrylamide is polymerized by acrylamide and methylene bisacrylamide according to a mass ratio of 29: 1.
The pH value of the separation gel is 8.8, and the pH value of the concentrated gel is 6.8.
Specifically, the colorant is processed Direct red 80, wherein the Direct red 80 is processed as follows:
(1) dissolving powder Direct red 80 in ultrapure water, and centrifuging to obtain an upper layer solution;
(2) sequentially carrying out activated carbon adsorption and gelatin adsorption, and centrifuging to obtain a supernatant solution;
(3) and (4) freeze-drying the supernatant to obtain a solid which is the finally required colorant.
The invention also provides a preparation method of the colored polyacrylamide gel, which comprises the following steps:
(1) preparing a stock solution: the stock solution comprises 30% of Acr-Bis, 4 multiplied by upper layer glue buffer solution, 4 multiplied by lower layer glue buffer solution and ammonium persulfate solution;
(2) preparing polyacrylamide separation gel: mixing 30% Acr-Bis, 0.1% SDS, 0.375M Tris-HCl, 0.1% tetraethyl ethylenediamine and 0.002% -0.01% coloring agent, adding ammonium persulfate, mixing, injecting into the gap between two glass plates, adding alcohol or water to cover on the separation gel;
(3) preparing polyacrylamide concentrated gel: taking 30 percent of Acr-Bis, 0.25M Tris-HCl, 0.1 percent of SDS, 0.1 percent of tetraethylethylenediamine and 0.02 to 0.1 percent of coloring agent according to the formula amount, fully and uniformly mixing, adding ammonium persulfate, fully and uniformly mixing, pouring out the liquid of the covering layer after the separation gel is completely polymerized, washing with deionized water, then pouring the prepared concentrated gel on the separation gel, immediately inserting a clean matched comb to avoid bubbles, and pulling out the comb after the concentrated gel is polymerized to form a sample adding hole.
The invention further provides a color polyacrylamide gel kit, which consists of 30% of Acr-Bis, 4 multiplied by upper layer gel buffer solution, 4 multiplied by lower layer gel buffer solution and ammonium persulfate, wherein the 4 multiplied by upper layer gel buffer solution consists of 0.25M Tris-HCl, 0.1% SDS, 0.1% tetraethylethylenediamine and 0.02% -0.1% of colorant, and the 4 multiplied by lower layer gel buffer solution consists of 0.375M Tris-HCl, 0.1% SDS, 0.1% tetraethylethylenediamine and 0.002% -0.01% of colorant.
Wherein the colorant is processed Direct red 80, wherein the Direct red 80 is processed as follows:
(1) dissolving powder Direct red 80 in ultrapure water, and centrifuging to obtain an upper layer solution;
(2) sequentially carrying out activated carbon adsorption and gelatin adsorption, and centrifuging to obtain a supernatant solution;
(3) and (4) freeze-drying the supernatant to obtain a solid which is the finally required colorant.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the accelerator is premixed in the 4 multiplied upper layer gel buffer solution and the 4 multiplied lower layer gel buffer solution, so that the design of reducing the toxicity of the accelerator ensures that experimenters are not damaged by volatile toxic compounds;
(2) the colorant is directly added into the 4 multiplied upper layer gel buffer solution and the 4 multiplied lower layer gel buffer solution, so that a user can conveniently judge whether the concentrated gel and the separation gel are solidified and loaded, and the protein separation effect is not influenced;
(3) and the glue preparation reagents are mixed in advance, so that the glue preparation process is accelerated, and the experiment efficiency is improved.
Drawings
FIG. 1 is a 10% colored polyacrylamide gel prepared in example 3, with the upper layer being overlying alcohol or water and the lower layer being a light red separating gel (not solidified);
FIG. 2A is a 10% colored polyacrylamide gel prepared in example 3, with the upper layer being a red concentrated gel (uncured) and the lower layer being a colorless isolated gel (cured);
FIG. 2B is a 10% colored polyacrylamide gel prepared in example 3, with the upper layer being a pink concentrated gel (set) and the lower layer being a colorless isolated gel (set);
FIG. 3 is a 10% color polyacrylamide gel prepared in example 3 after electrophoresis with a Marker added thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample;
FIG. 4 is a graph of a 10% polyacrylamide gel prepared in comparative example 1 after electrophoresis with a Marker added thereto and a protein sample, lane 1 being the Marker, lane 2 being the protein sample;
FIG. 5 is a graph of 6% colored polyacrylamide gel prepared in example 1 after electrophoresis with Marker and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample;
FIG. 6 is a graph of an 8% colored polyacrylamide gel prepared in example 2 after electrophoresis with a Marker added thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample;
FIG. 7 is a 12% color polyacrylamide gel prepared in example 4 after electrophoresis with a Marker added thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample;
FIG. 8 is a 15% colored polyacrylamide gel prepared in example 5 after electrophoresis with a Marker attached thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.
The Marker used in the examples is a pre-dyed color protein molecular weight Marker of Kyowa Kayji Biotechnology GmbH, cat # KGM 471; the protein sample was Bovine Serum Albumin (BSA).
According to different optimal separation ranges required by different protein samples, experimenters generally select gels with different concentrations to meet the experimental requirements, and the invention can prepare the gels within the concentration range of 6-15%.
Example 16% colored polyacrylamide gel formulation.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml, 1M Tris-HCl, 0.4% SDS, 0.1% tetraethyl ethylenediamine and 0.02% -0.1% colorant, pH 6.8;
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4 percent of SDS, 0.4 percent of tetraethyl ethylenediamine and 0.002 to 0.01 percent of colorant, wherein the pH value is 8.8;
4) ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
Preparation method of 2.6% color polyacrylamide separation gel
2.7ml of distilled water, 1.0ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into a gap between two glass plates, and water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully mixing, adding 0.02ml of 10% ammonium persulfate, fully mixing, pouring out the covering layer liquid after the separation gel is changed from light red to colorless, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and after the concentrated gel is changed from red to pink, pulling out a comb to form a sample adding hole, thus carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis experiment.
FIG. 5 is a 6% color polyacrylamide gel prepared in example 1 after electrophoresis with a Marker added thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample.
Example 28% color polyacrylamide gel formulation.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml, 1M Tris-HCl, 0.4% SDS, 0.1% tetraethyl ethylenediamine and 0.02% -0.1% colorant, pH 6.8;
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4 percent of SDS, 0.4 percent of tetraethyl ethylenediamine and 0.002 to 0.01 percent of colorant, wherein the pH value is 8.8;
4) ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
2. Preparation method of 8% colored polyacrylamide separation gel
2.4ml of distilled water, 1.3ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into the gap between the two glass plates, and then water is added to cover the separation gel.
3. Preparation method of 5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully mixing, adding 0.02ml of 10% ammonium persulfate, fully mixing, pouring out the covering layer liquid after the separation gel is changed from light red to colorless, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and after the concentrated gel is changed from red to pink, pulling out a comb to form a sample adding hole, thus carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis experiment.
FIG. 6 is a graph of the 8% colored polyacrylamide gel prepared in example 2 after electrophoresis with Marker added to the protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample.
Example 310% color polyacrylamide gel configuration.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml, 1M Tris-HCl, 0.4% SDS, 0.1% tetraethylethylenediamine and 0.02% -0.1% colorant, pH 6.8
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4% SDS, 0.4% tetraethylethylenediamine and 0.002% -0.01% coloring agent, pH 8.8
4) Ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
Preparation method of 2.10% color polyacrylamide separation gel
2.0ml of distilled water, 1.7ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into the gap between the two glass plates, and then water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully mixing, adding 0.02ml of 10% ammonium persulfate, fully mixing, pouring out the covering layer liquid when the separation gel is changed from light red to colorless, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and pulling out a comb to form a sample adding hole when the concentrated gel is changed from red to pink, thus carrying out SDS-PAGE electrophoresis experiment.
FIG. 1 is a 10% colored polyacrylamide gel prepared in example 6, with the upper layer being overlying alcohol or water and the lower layer being a light red separating gel (not solidified);
FIG. 2A is a 10% colored polyacrylamide gel prepared in example 6, with the upper layer being a red concentrated gel (uncured) and the lower layer being a colorless isolated gel (cured);
FIG. 2B shows a 10% colored polyacrylamide gel prepared in example 6, with a red concentrated gel (solidified) on the top layer and a colorless isolated gel (solidified) on the bottom layer.
Thus, the color change of the separation gel and the concentration gel can be directly used to judge whether the gel is solidified and when the sample should be added.
Example 412% color polyacrylamide gel configuration.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml, 1M Tris-HCl, 0.4% SDS, 0.1% tetraethyl ethylenediamine and 0.02% -0.1% colorant, pH 6.8;
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4 percent of SDS, 0.4 percent of tetraethyl ethylenediamine and 0.002 to 0.01 percent of colorant, wherein the pH value is 8.8;
4) ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
Preparation method of 2.12% colored polyacrylamide separation gel
1.7ml of distilled water, 2.0ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into a gap between two glass plates, and water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully and uniformly mixing, adding 0.02ml of 10% ammonium persulfate, fully and uniformly mixing, pouring out the covering layer liquid after the separation gel turns into colorless with light red, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and after the concentrated gel turns into pink from red, pulling out a comb to form a sample adding hole, thus carrying out SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis experiment.
FIG. 7 is a 12% color polyacrylamide gel prepared in example 4 after electrophoresis with Marker added and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample.
Example 515% color polyacrylamide gel configuration.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml, 1M Tris-HCl, 0.4% SDS, 0.1% tetraethylethylenediamine and 0.02% -0.1% colorant, pH 6.8
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4% SDS, 0.4% tetraethylethylenediamine and 0.002% -0.01% coloring agent, pH 8.8
4) Ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
Preparation method of 2.15% colored polyacrylamide separation gel
1.2ml of distilled water, 2.5ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into a gap between two glass plates, and water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully mixing, adding 0.02ml of 10% ammonium persulfate, fully mixing, pouring out the covering layer liquid after the separation gel is changed from light red to colorless, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and after the concentrated gel is changed from red to pink, pulling out a comb to form a sample adding hole, thus carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis experiment.
FIG. 8 is a 15% colored polyacrylamide gel prepared in example 5 after electrophoresis with a Marker attached thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample.
Example 610% color native polyacrylamide gel configuration.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29 g of acrylamide and 1 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)4 × supernatant buffer: 30ml of 1M Tris-HCl, 0.1 percent of tetraethyl ethylenediamine and 0.02 to 0.1 percent of colorant, and the pH value is 6.8
3)4 × lower layer gel buffer: 30ml of 1.5M Tris-HCl, 0.4 percent tetraethylethylenediamine and 0.002 to 0.01 percent of colorant, and the pH value is 8.8
4) Ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is removed, 1ml of water is added for dissolving and mixing evenly.
Preparation method of 2.10% color non-denatured polyacrylamide separation gel
2.0ml of distilled water, 1.7ml of 30 percent Acr-Bis and 1.25ml of 4 multiplied lower layer gel buffer solution are taken, fully and evenly mixed, 50ul of 10 percent ammonium persulfate is added, fully and evenly mixed and rapidly injected into the gap between the two glass plates, and then water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.17ml of distilled water, 0.33ml of 30% Acr-Bis and 0.5ml of 4 multiplied upper layer gel buffer solution, fully mixing, adding 0.02ml of 10% ammonium persulfate, fully mixing, pouring out the covering layer liquid after the separation gel is changed from light red to colorless, then pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and after the concentrated gel is changed from red to pink, pulling out a comb to form a sample adding hole, thus carrying out the next polyacrylamide gel electrophoresis experiment.
The concentration of acrylamide and methylene acrylamide in the solution is determined based on other concentrations of 6-15%, so that the non-denaturing polyacrylamide gel in the concentration range can be realized according to the formula of the invention, and the specific steps are the same.
Comparative example 110% polyacrylamide gel formulation.
1. Each mixed stock solution component:
1) 30% Acr-Bis: 100ml, taking 29.2 g of acrylamide and 0.8 g of methylene bisacrylamide, adding water to dissolve, and fixing the volume to 100ml and mixing uniformly;
2)2MTris-Hcl buffer: 100ml, taking 24.2228 g of Tris, adding a proper amount of water for dissolution, adjusting the pH value to 8.8 by using Hcl, metering to 100ml and mixing uniformly.
3)1MTris-Hcl buffer: 50ml, taking 6.057 g of Tris, adding a proper amount of water for dissolution, adjusting the pH value to 6.8 by using Hcl, metering to 50ml and mixing uniformly.
4) 10% SDS: 5ml, taking 0.5 g of SDS, adding water for dissolving, and fixing the volume to 5 ml.
4) Ammonium persulfate: 10 percent, 0.1 g of ammonium persulfate is taken and dissolved in 1ml of water to be mixed evenly.
Preparation method of 2.10% polyacrylamide separation gel
2.2ml of distilled water, 1.7ml of 30% Acr-Bis, 1ml of 2M Tris-HCl buffer solution and 50ul of 10% SDS are taken and mixed uniformly, then 50ul of 10% ammonium persulfate is added and mixed uniformly, then 2ul of TEMED is added and mixed uniformly, and then the mixture is injected into the gap between the two glass plates, and then alcohol or water is added to cover the separation gel.
Preparation method of 3.5% polyacrylamide concentrated gel
Taking 1.4ml of distilled water, 0.33ml of 30% Acr-Bis, 250ul of 1M Tris-HCl buffer solution and 50ul of 10% SDS, fully mixing, adding 20ul of 10% ammonium persulfate, fully mixing, adding 2ul of TEMED, pouring out covering layer liquid after separation gel is completely polymerized, pouring the prepared concentrated gel on the separation gel, immediately inserting clean matched trees to avoid bubbles, and pulling out a comb to form a sample adding hole after the concentrated gel is polymerized, thus carrying out the next polyacrylamide gel electrophoresis experiment.
FIG. 4 is a graph of a 10% polyacrylamide gel prepared in comparative example 1 after electrophoresis with a Marker added thereto and a protein sample, wherein lane 1 is the Marker and lane 2 is the protein sample. From the separation effect of the Marker and the protein sample shown in fig. 3 and 4, the polyacrylamide gel prepared by the present invention has substantially the same separation effect as the conventional polyacrylamide gel.
The separation gel disclosed by the invention contains 0.002-0.01% of coloring agent, so that the color of the separation gel is changed before and after solidification, the solidification degree of the separation gel is conveniently judged, the concentrated gel is conveniently prepared in time, and the separation effect of protein is not influenced; the concentrated glue contains 0.02-0.1% of colorant, so that the color of the concentrated glue changes before and after solidification, the solidification degree of the concentrated glue can be conveniently judged, and a user can conveniently sample the concentrated glue without influencing the separation effect of the protein.
The present invention provides a method for preparing colored polyacrylamide gel, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Claims (7)
1. The variable-color polyacrylamide gel is characterized by comprising separation gel and concentration gel, wherein the separation gel comprises 6-15% of polyacrylamide, 0.375M Tris-HCl, 0.1% of SDS, 10% of ammonium persulfate, 0.1% of tetraethylethylenediamine and 0.002-0.01% of colorant; the concentrated gel comprises 5% of polyacrylamide, 0.25M Tris-HCl, 0.1% of SDS, 0.1% of ammonium persulfate, 0.1% of tetraethylethylenediamine and 0.02% -0.1% of colorant.
2. The color-changeable polyacrylamide gel according to claim 1 wherein the polyacrylamide is polymerized from acrylamide and methylene bisacrylamide in a mass ratio of 29: 1.
3. The color-changeable polyacrylamide gel of claim 1 wherein the pH of said separation gel is 8.8 and the pH of said concentration gel is 6.8.
4. The color-changeable polyacrylamide gel of claim 1 wherein the colorant is treated Direct red 80, wherein Direct red 80 is treated by:
(1) dissolving powder Direct red 80 in ultrapure water, and centrifuging to obtain an upper layer solution;
(2) sequentially carrying out activated carbon adsorption and gelatin adsorption, and centrifuging to obtain a supernatant solution;
(3) and (4) freeze-drying the supernatant to obtain a solid which is the finally required colorant.
5. The method for preparing colored polyacrylamide gel according to any one of claims 1-4, comprising the steps of:
(1) preparing a stock solution: the stock solution comprises 30% of Acr-Bis, 4 multiplied by upper layer glue buffer solution, 4 multiplied by lower layer glue buffer solution and ammonium persulfate solution;
(2) preparing polyacrylamide separation gel: taking 30 percent of Acr-Bis, 0.1 percent of SDS, 0.375M of Tris-HCl, 0.1 percent of tetraethyl ethylenediamine and 0.002 to 0.01 percent of colorant according to the formula amount, fully mixing, adding ammonium persulfate, fully mixing, then quickly injecting into a gap between two glass plates, and then adding alcohol or water to cover on separation gel;
(3) preparing polyacrylamide concentrated gel: taking 30% of Acr-Bis, 0.25M Tris-HCl, 0.1% of SDS, 0.1% of tetraethylethylenediamine and 0.02% -0.1% of coloring agent according to the formula amount, fully and uniformly mixing, adding ammonium persulfate, fully and uniformly mixing, pouring out the covering layer liquid after the separation gel is completely polymerized, washing with deionized water, pouring the prepared concentration gel on the separation gel, immediately inserting a clean matched comb to avoid bubbles, and pulling out the comb after the concentration gel is polymerized to form a sample adding hole.
6. The color polyacrylamide gel kit is characterized by comprising 30% of Acr-Bis, 4 multiplied by upper layer gel buffer solution, 4 multiplied by lower layer gel buffer solution and ammonium persulfate, wherein the 4 multiplied by upper layer gel buffer solution comprises 0.25M Tris-HCl, 0.1% SDS, 0.1% tetraethylethylenediamine and 0.02% -0.1% of colorant, and the 4 multiplied by lower layer gel buffer solution comprises 0.375M Tris-HCl, 0.1% SDS, 0.1% tetraethylethylenediamine and 0.002% -0.01% of colorant.
7. The kit of claim 6, wherein the staining agent is a processed Direct red 80, wherein Direct red 80 is processed by:
(1) dissolving powder Direct red 80 in ultrapure water, and centrifuging to obtain an upper layer solution;
(2) sequentially carrying out activated carbon adsorption and gelatin adsorption, and centrifuging to obtain a supernatant solution;
(3) and (4) freeze-drying the supernatant to obtain a solid which is the finally required colorant.
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| US6190522B1 (en) * | 1998-04-24 | 2001-02-20 | Board Of Regents, The University Of Texas System | Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis |
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| CN108181369A (en) * | 2017-12-22 | 2018-06-19 | 重庆康萃医药科技有限公司 | A kind of polyacrylamide gel and its kit |
| CN108627564A (en) * | 2018-05-08 | 2018-10-09 | 苏州勇泰生物科技有限公司 | A kind of polyacrylamide gel of efficient stable and preparation method thereof |
| CN110527017A (en) * | 2019-09-16 | 2019-12-03 | 武汉赛维尔生物科技有限公司 | A kind of polyacrylamide gel and preparation method thereof for small molecular weight protein separation |
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2020
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| US6190522B1 (en) * | 1998-04-24 | 2001-02-20 | Board Of Regents, The University Of Texas System | Analysis of carbohydrates derivatized with visible dye by high-resolution polyacrylamide gel electrophoresis |
| CN105021820A (en) * | 2014-11-17 | 2015-11-04 | 中国农业大学 | Polyacrylamide gel and application of same in electrophoretic separation of small molecular protein or polypeptide |
| CN108181369A (en) * | 2017-12-22 | 2018-06-19 | 重庆康萃医药科技有限公司 | A kind of polyacrylamide gel and its kit |
| CN108627564A (en) * | 2018-05-08 | 2018-10-09 | 苏州勇泰生物科技有限公司 | A kind of polyacrylamide gel of efficient stable and preparation method thereof |
| CN110527017A (en) * | 2019-09-16 | 2019-12-03 | 武汉赛维尔生物科技有限公司 | A kind of polyacrylamide gel and preparation method thereof for small molecular weight protein separation |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114235933A (en) * | 2021-12-22 | 2022-03-25 | 安徽昊拓生物科技有限公司 | Dye of colored polyacrylamide gel and preparation method thereof |
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