Dyeing-free gradient gel and preparation method thereof
By preparing stain-free gradient gels, the problems of poor repeatability of polyacrylamide gel electrophoresis, invasion of toxic reagents and inadequate automated production in the prior art are solved, and efficient and clear protein separation and simplified operation process are achieved.
Patent Information
- Application Number
- CN202510518766.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-07-29
AI Technical Summary
The existing polyacrylamide gel electrophoresis technology has problems such as poor repeatability, toxic reagent invasion, complex production, high cost, and unsuitable for automated production and long-term dyeing after electrophoresis.
Automatic glue filling equipment is used to prepare stain-free gradient gels, and gradient gels composed of Tris-HCl buffer, acrylamide, sodium dodecyl sulfate, ammonium persulfate solution, glycerol and tetramethylethylenediamine are used to achieve one-step preparation. After electrophoresis, clear protein bands can be presented at 300nm ultraviolet.
It achieves efficient and clear protein separation, avoids the use of toxic reagents, is suitable for automated production, simplifies operating procedures, and reduces health risks and environmental pollution.
Smart Images
Figure CN120383702A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of protein separation in the field of bioelectrophoresis, and specifically relates to a gradient stain-free polyacrylamide gel. Background Art
[0002] Polyacrylamide gel electrophoresis, also known as PAGE (polyacrylamide gel electrophoresis), is an electrophoresis technique using polyacrylamide gel as the supporting medium, and is commonly used for the separation of mixed protein samples. The commonly used SDS-PAGE gel in the laboratory is a discontinuous polyacrylamide gel system, which is divided into two components: a stacking gel and a separating gel. During electrophoresis, the stacking gel is located in the upper layer of the vertical electrophoresis system and has a concentrating effect. The separating gel is located in the lower layer. After the protein mixed sample enters the separating gel, it will be separated according to the molecular weight of the protein itself. This technique is currently the most effective means for the separation and detection of protein samples in the laboratory.
[0003] Currently, the commonly used protein gels are still manually made, which have problems such as poor repeatability and being vulnerable to toxic reagents.
[0004] In conventional experiments, in order to obtain better experimental results, researchers need to make protein gels with different concentrations according to the molecular weight of the protein, which increases the workload. Since the gradient gel itself has multiple concentrations, it can adapt to proteins of different molecular weights. Currently, the gradient gels mainly come from commercial products, which are relatively expensive and the separation effect is not good enough. Moreover, the existing formulations are difficult to stably produce gradient gels, and are not suitable for automated production of gradient gels. In addition, after electrophoresis, Coomassie Brilliant Blue R-250 or G-250 is required for staining, and then a decolorizing solution containing organic substances such as methanol and acetic acid is used for repeated elution to observe the bands. The staining and repeated decolorization are time-consuming, about 2 - 4 hours. Moreover, both methanol and acetic acid are hazardous chemicals, and there are problems such as difficult procurement. The methanol and acetic acid mixed solution that the experimenter needs to contact repeatedly not only has a strong pungent smell but also has toxicity, seriously damaging the health of the experimenter. At the same time, the organic waste generated also poses a non-negligible hazard to the environment. Summary of the Invention
[0005] Based on this, the present application provides a stain-free gradient gel and its preparation method. The separation effect of this gradient gel is good, and its formulation can achieve stable and automated production of stain-free gradient gels.
[0006] The specific technical solutions are as follows:
[0007] This application provides a stain-free gradient gel. The gradient gel includes two separating gels with different concentrations, namely the first separating gel and the second separating gel. The components are: Tris-HCl buffer, acrylamide mixed solution, trihalide compound, sodium dodecyl sulfate solution, ammonium persulfate solution, glycerol, tetramethylethylenediamine, and water. An automated gel casting device is used for gel casting.
[0008] In one embodiment, the gradient gel includes the first separating gel and the second separating gel. Among them, the first separating gel includes the following volume content ratios: 1.2 - 5.0 parts of Tris-HCl buffer, 2.0 - 6.0 parts of acrylamide mixed solution, 0.025 - 0.1 part of trihalide compound, 0.05 - 0.2 part of sodium dodecyl sulfate solution, 0.5 - 0.2 part of ammonium persulfate solution, 0.5 - 2.0 parts of glycerol, 0.004 - 0.008 part of tetramethylethylenediamine, and 1.0 - 10.0 parts of water. The volume content ratios of each component in the second separating gel are as follows: 1.2 - 5.0 parts of Tris-HCl buffer, 2.0 - 6.0 parts of acrylamide mixed solution, 0.025 - 0.1 part of trihalide compound, 0.05 - 0.2 part of sodium dodecyl sulfate solution, 0.5 - 0.2 part of ammonium persulfate solution, 0.5 - 2.0 parts of glycerol, 0.004 - 0.008 part of tetramethylethylenediamine, and 1.0 - 10.0 parts of water. Among them, the concentration of polyacrylamide in the first separating gel > the concentration of polyacrylamide in the second separating gel.
[0009] This application provides a preparation method of the gradient gel described above. The method includes the following steps:
[0010] Prepare the corresponding gel solutions according to the components of the first separating gel and the second separating gel in the gradient gel; pour the gel solutions of the first separating gel and the second separating gel into the gel plate in sequence, insert the comb, and wait for the gel to solidify to obtain the gradient gel.
[0011] The glycerol concentration ratio in the first separating gel and the second separating gel described in this application is (10 - 5):(3 - 2).
[0012] The volume ratio of the first separating gel and the second separating gel described in this application is (2 - 5):(5 - 2).
[0013] In the separating gel described in this application, the Tris concentration in the Tris-HCl buffer is 1.45 - 1.55 mol / L, and the pH is adjusted to 8.7 - 8.9 with hydrochloric acid.
[0014] The trihalide compound described in this application is selected from at least one of chloroform, trichloroacetic acid, and trichloroethanol.
[0015] For the SDS-PAGE polyacrylamide gel of some examples of the present invention, by using a trihalide to bind to tryptophan and under the photoactivation of 300 nm ultraviolet light, the protein bands containing tryptophan can emit light in the gel. After electrophoresis, clear protein bands can be presented by irradiating with 300 nm ultraviolet light for 60 - 90 s. It overcomes the problems that after traditional SDS-PAGE electrophoresis, Coomassie Brilliant Blue staining is required for more than 30 minutes, and repeated decolorization with organic reagents such as methanol and acetic acid is needed after staining. It overcomes the influence of Coomassie Brilliant Blue staining from different manufacturers and the decolorization degree of the decolorizing solution on the detection sensitivity, and also avoids the health hazards to experimental personnel caused by using toxic and irritating organic substances such as methanol and acetic acid.
[0016] Compared with the traditional technology, the stain-free gradient gel provided by the present application has the following beneficial effects:
[0017] The present invention has the following beneficial effects:
[0018] First, the gradient gel provided by the present application has the advantages of being stain-free, having clear bands, good separation effect, and low toxicity.
[0019] Second, the optimized formula of the present application is more suitable for the preparation of gradient gels. The combination of components in the formula makes it more suitable for automated gel-casting equipment, realizing the automated production of gradient gels and creating value. Description of the Drawings
[0020] Figure 1 It is a stain-free gradient (4 - 15%) polyacrylamide gel. Detailed Embodiments
[0021] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the present invention will be further described below with reference to specific illustrations.
[0022] Example 1 Preparation of 4% - 15% gradient gel
[0023] This example provides a 4% - 15% gradient gel, which includes high- and low-concentration gels.
[0024] The concentration of the high-concentration gel is 15%. The volume content ratio of each component: 8 parts of Tris-HCl buffer, 5 parts of acrylamide mixed solution, 0.05 part of trihalide, 0.1 part of sodium dodecyl sulfate solution, 0.3 part of ammonium persulfate solution, 1 part of glycerol, 0.006 part of tetramethylethylenediamine, and 6 parts of water. Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.
[0025] The concentration of the low-concentration gel is 4%. The volume content ratio of each component is as follows: 8 parts of Tris-HCl buffer, 2 parts of acrylamide mixed solution, 0.05 part of trihalide compound, 0.1 part of sodium dodecyl sulfate solution, 0.3 part of ammonium persulfate solution, 1 part of glycerol, 0.006 part of tetramethylethylenediamine, and 6 parts of water. Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.
[0026] The preparation method of the above 4%-15% gradient gel includes the following steps:
[0027] (1) Prepare high- and low-concentration gels:
[0028] 15% high-concentration gel: Prepare the required volume of gel solution according to the above formula.
[0029] 4% low-concentration gel: Prepare the required volume of gel solution according to the above formula.
[0030] (2) Connect the high-concentration gel solution (15%) to pump 1 of the automatic gel-casting equipment, and connect the medium-concentration gel solution (4%) to pump 2.
[0031] (3) Set the automatic program. The program content is that pump 1 starts to pump 5 ml of high-concentration gel solution into the gel plate (volume 10 ml); immediately afterwards, pump 1 pauses, pump 2 starts, and pumps 5 ml of low-concentration gel solution into the gel plate, and the pump starts until the gel plate is filled.
[0032] (4) Turn on the automatic gel-casting instrument to complete the gel casting. Manual gel casting can also be carried out in the above manner.
[0033] After the gel preparation is completed, it is compared with the commercially available product a of the same specification (ACE Biotechnology, product number: ET12412Gel) by PAGE electrophoresis. It is found that the electrophoresis bands of the gradient gel provided in this example are clear, rich in details, and the overall effect is excellent.
[0034] Example 2 Preparation of 4%-12% gradient gel
[0035] This example provides a 4%-12% gradient gel, which includes high- and low-concentration gels.
[0036] The concentration of the high-concentration gel is 12%. The volume content ratio of each component is as follows: 8 parts of Tris-HCl buffer, 5 parts of acrylamide mixed solution, 0.05 part of trihalide compound, 0.1 part of sodium dodecyl sulfate solution, 0.3 part of ammonium persulfate solution, 1 part of glycerol, 0.006 part of tetramethylethylenediamine, and 6 parts of water. Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.
[0037] The concentration of the low-concentration gel is 4%. The volume content ratio of each component is as follows: 8 parts of Tris-HCl buffer solution, 2 parts of acrylamide mixed solution, 0.05 part of trihalide compound, 0.1 part of sodium dodecyl sulfate solution, 0.3 part of ammonium persulfate solution, 1 part of glycerol, 0.006 part of tetramethylethylenediamine, and 6 parts of water. Among them, polyacrylamide is prepared from acrylamide and methylene bisacrylamide, and the mass ratio of acrylamide to methylene bisacrylamide is 29:1.
[0038] The preparation method of the above-mentioned 4%-12% gradient gel includes the following steps:
[0039] (1) Prepare high- and low-concentration gels:
[0040] 12% high-concentration gel: Prepare the required volume of gel solution according to the above formula.
[0041] 4% low-concentration gel: Prepare the required volume of gel solution according to the above formula.
[0042] (2) Connect the high-concentration gel solution (12%) to pump 1 of the automatic gel filling device, and connect the medium-concentration gel solution (4%) to pump 2.
[0043] (3) Set the automatic program. The program content is that pump 1 starts to pump 5 ml of high-concentration gel solution into the gel plate (volume 10 ml); immediately afterwards, pump 1 pauses, pump 2 starts, and pumps 5 ml of low-concentration gel solution into the gel plate, and the pump starts until the gel plate is filled.
[0044] (4) Turn on the automatic gel filling instrument to complete the gel filling. Manual gel filling can also be carried out in the above manner.
[0045] The above is only the preferred implementation mode of the present invention. The protection scope of the present invention is not limited to the above implementation mode. Any technical solution belonging to the principle of the present invention belongs to the protection scope of the present invention. For those skilled in the art, several improvements made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.
Claims
1. A stain-free gradient gel and its preparation method, characterized in that, The gradient gel includes a first separating gel and a second separating gel. Among them, the first separating gel includes the following volume content ratios: 1.2 - 5.0 parts of Tris-HCl buffer solution, 2.0 - 6.0 parts of acrylamide mixed solution, 0.025 - 0.1 part of trihalide compound, 0.05 - 0.2 part of sodium dodecyl sulfate solution, 0.5 - 0.2 part of ammonium persulfate solution, 0.5 - 2.0 parts of glycerol, 0.004 - 0.008 part of tetramethylethylenediamine, and 1.0 - 10.0 parts of water; the volume content ratios of each component in the second separating gel are as follows: 1.2 - 5.0 parts of Tris-HCl buffer solution, 2.0 - 6.0 parts of acrylamide mixed solution, 0.025 - 0.1 part of trihalide compound, 0.05 - 0.2 part of sodium dodecyl sulfate solution, 0.5 - 0.2 part of ammonium persulfate solution, 0.5 - 2.0 parts of glycerol, 0.004 - 0.008 part of tetramethylethylenediamine, and 1.0 - 10.0 parts of water; among them, the concentration of polyacrylamide in the first separating gel > the concentration of polyacrylamide in the second separating gel; an automated gel filling device is used for gel filling.
2. The stain-free gradient gel according to claim 1 and its preparation method, characterized in that, The glycerol concentration ratio in the first separating gel and the second separating gel is (10 - 5):(3 - 2).
3. The stain-free gradient gel according to claim 1 and its preparation method are characterized in that, The volume ratio of the first separating gel and the second separating gel is (2 - 5):(5 - 2).
4. The stain-free gradient gel according to claim 1 and its preparation method, characterized in that, In the separating gel, the Tris concentration in the Tris-HCl buffer solution is 1.45 - 1.55 mol / L, and the pH is adjusted to 8.7 - 8.9 with hydrochloric acid.
5. The stain-free gradient gel according to claim 1 and its preparation method are characterized in that, The trihalide compound is selected from at least one of chloroform, trichloroacetic acid, and trichloroethanol.
6. The stain-free gradient gel according to claim 1 and its preparation method are characterized in that, An appropriate amount of acid dye is also added to the gel.
7. The non-staining gradient gel according to claim 6 and its preparation method, characterized in that, The acid dye is selected from at least one of tartrazine, brilliant blue FCF, or new carmine.