Gel plate for detecting GLP-1 analogue, preparation method and method for detecting GLP-1 analogue
By designing electrophoresis gel plates with specific structures and using precise electrophoresis methods, the problem of poor separation of GLP-1 analogs in existing technologies has been solved, achieving clear separation and accurate quantitative detection of GLP-1 analogs.
Patent Information
- Application Number
- CN202511316897.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-16
- Publication Date
- 2025-12-12
AI Technical Summary
Existing SDS-PAGE electrophoresis methods cannot effectively separate and detect GLP-1 analogs with molecular weights in the range of 3-7 kDa, especially when impurities with smaller molecular weights are present, as they cannot form clear detection bands.
An electrophoretic gel plate was designed, comprising a stacking gel layer, a separator gel layer, and a separating gel layer. By controlling the concentration and length of the acrylamide mixture, a pore structure suitable for GLP-1 analogs was formed. Combined with electrophoresis, fixation, staining, and destaining treatments, accurate detection of GLP-1 analogs was achieved.
Clear separation and accurate quantitative detection of GLP-1 analogs were achieved, and well-defined bands were formed in the 3-7KD region, solving the problem of poor detection performance in existing technologies.
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Figure CN121114181A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of protein detection technology, and in particular to a gel plate for detecting GLP-1 analogs, a method for preparing a gel plate for detecting GLP-1 analogs, and a method for detecting GLP-1 analogs. Background Technology
[0002] Glucagon-like peptide-1 (GLP-1) is a family of incretins secreted by intestinal cells. It is a hormone secreted after meals and helps control blood sugar. Specifically, its main metabolic reactions in the human body are: release in response to the increase in postprandial blood glucose deionization levels, stimulating insulin production; inhibiting glucagon release; and slowing the rate at which nutrients are absorbed into the bloodstream. However, because GLP-1 is rapidly inactivated and degraded in the human body by an enzyme (DPP-4), it cannot be directly used as a drug to control blood sugar.
[0003] Furthermore, glucagon-like peptide-1 (GLP-1) analogs are also GLP-1 receptor agonists, exhibiting similar activity to GLP-1 and a high affinity for the GLP-1 receptor. Both can mimic natural gastrointestinal hormones in the human body, promoting insulin secretion and regulating blood glucose. Currently, the main production method for GLP-1 analogs involves obtaining the amino acid backbone through fermentation, followed by chemical coupling to link the side chains, thus obtaining the complete GLP-1 analog.
[0004] Glucagon-like peptide-1 (GLP-1) analogs, such as liraglutide, smegglutide, and semaglutide, have molecular weights of approximately 3-7 kDa. Current SDS-PAGE protein electrophoresis methods are not effective at separating GLP-1 analogs. When GLP-1 analogs contain other impurities with smaller molecular weights, they cannot form clear bands that are distinct from the other impurities and suitable for detection. Summary of the Invention
[0005] To address the problems in the prior art, the present invention provides the following solution.
[0006] On one hand, the present invention discloses an electrophoretic gel plate for detecting the content of GLP-1 analogs. The electrophoretic gel plate comprises, in sequence along the electrophoresis direction, a stacking gel layer, a separator gel layer, and a separating gel layer. The concentration of the acrylamide mixture in the separating gel layer is 0.15-0.17 g / ml, and the length of the separating gel layer along the electrophoresis direction is greater than 5 cm. The concentration of the acrylamide mixture in the separator gel layer is 0.08-0.12 g / ml, and the length of the separator gel layer along the electrophoresis direction is 1-3 cm. The concentration of the acrylamide mixture in the stacking gel layer is 0.02-0.05 g / ml, and the length of the stacking gel layer along the electrophoresis direction is greater than 1 cm.
[0007] On one hand, this invention discloses a method for preparing an electrophoretic gel plate for detecting the content of GLP-1 analogs, comprising the following steps:
[0008] Acrylamide mixture, water, and gel buffer are mixed to form a separating gel preparation solution, a separator gel preparation solution, and a stacking gel preparation solution for preparing electrophoresis gel plates; the separating gel preparation solution with an acrylamide mixture content of 0.15-0.17 g / ml is added to the gel plate, and after solidification, a first transition gel plate with a separating gel layer having a length greater than 5 cm along the electrophoresis direction is formed.
[0009] The acrylamide mixture with a content of 0.08-0.12 g / ml is added to the first transition plate, and after solidification, a second transition plate with a separation layer with a length of 1-3 cm along the electrophoresis direction is formed.
[0010] The concentrated gel solution with an acrylamide mixture content of 0.02-0.05 g / ml is added to the second transition gel plate. After solidification, the electrophoresis gel plate is formed with a concentrated gel layer having a length greater than 1 cm along the electrophoresis direction. The concentrated gel layer of the electrophoresis gel plate has a sample loading port.
[0011] In one embodiment of the present invention, a method for preparing an electrophoretic gel plate for detecting the content of GLP-1 analogues includes: mixing acrylamide and methylenebisacrylamide in a mass ratio of (20-29):1 to obtain the acrylamide mixture, wherein the separating gel solution contains 4-6% glycerol.
[0012] In one embodiment of the present invention, the step of adding the separating gel preparation solution with an acrylamide mixture content of 0.15-0.17 g / ml to the gel preparation plate, and forming a first transition gel preparation plate with a separating gel layer having a length greater than 5 cm along the electrophoresis direction after solidification, includes: adding ammonium persulfate to the separating gel preparation solution at 0.4-0.6 ug / ml, and adding tetramethylethylenediamine to the separating gel preparation solution at 0.8-1.2 ug / ml to obtain the separating gel;
[0013] The separating gel is added to the gel-making plate, and after solidification, a first transition gel-making plate with a separating gel layer having a length of 6-9 cm along the electrophoresis direction is formed.
[0014] The step of adding the separator gel preparation solution with an acrylamide mixture content of 0.08-0.12 g / ml to the first transition gel preparation plate, and forming a second transition gel preparation plate with a separator gel layer having a length of 1-3 cm along the electrophoresis direction after solidification, includes: adding ammonium persulfate to the separator gel preparation solution at 0.7-0.9 ug / ml, and adding tetramethylethylenediamine to the separator gel preparation solution at 1.5-1.7 ug / ml to obtain the separator gel;
[0015] The separator adhesive is added to the first transition adhesive plate, and after solidification, a second transition adhesive plate is formed with a separator adhesive layer having a length of 1.5-2.5 cm along the electrophoresis direction.
[0016] The step of adding the concentrated gel preparation solution containing 0.02-0.05 g / ml of the acrylamide mixture to the second transition gel preparation plate, and forming the electrophoretic gel plate after solidification, comprising: adding ammonium persulfate to the concentrated gel preparation solution at 0.7-0.9 ug / ml, and adding tetramethylethylenediamine to the concentrated gel preparation solution at 1.5-1.7 ug / ml to obtain the concentrated gel;
[0017] The concentrated gel is added to the second transition gel plate, and after solidification, an electrophoretic gel plate is formed with a concentrated gel layer having a length of 2.5cm-4cm along the electrophoresis direction.
[0018] In one embodiment of the present invention, the step of adding the stacking gel to the second transition gel plate and solidifying it to form an electrophoresis gel plate having a stacking gel layer with a length of 2.5cm-4cm along the electrophoresis direction includes: pouring the stacking gel into the second transition gel plate and pouring it to the top of the second transition gel plate, inserting an electrophoresis comb and placing it until the stacking gel solidifies, and removing the electrophoresis comb to obtain the electrophoresis gel plate having the sample loading port.
[0019] In another aspect, the present invention discloses a method for detecting GLP-1 analogs, comprising: adding a sample to be tested to the aforementioned electrophoresis gel plate or an electrophoresis gel plate prepared according to the aforementioned method for preparing an electrophoresis gel plate for detecting GLP-1 analog content, and performing electrophoresis in an electrophoresis tank to obtain an electrophoretic protein gel; wherein the electrophoretic protein gel has a separation detection band, and the molecular weight of the GLP-1 analog is 3KD-7KD.
[0020] In one embodiment of the present invention, obtaining the electrophoretic protein gel after electrophoresis in an electrophoresis tank includes: installing the electrophoresis gel plate into the inner tank of the electrophoresis tank, adding a negative electrophoresis buffer containing 0.05-0.15 mol / L tris(hydroxymethyl)aminomethane to the inner tank, and adding a positive electrophoresis buffer containing 0.15-0.25 mol / L tris(hydroxymethyl)aminomethane to the outer tank of the electrophoresis tank.
[0021] In one embodiment of the present invention, the electrophoresis gel plate includes a staining agent; the process of obtaining the electrophoretic protein gel after electrophoresis in the electrophoresis tank further includes: connecting the electrophoresis tank to a power source, electrophoresing the electrophoresis gel plate at an electric field strength of 3-5V / cm for 10-15 minutes, and then electrophoresing at an electric field strength of 8V / cm-15V / cm until the staining agent reaches the bottom of the electrophoresis gel plate, and then stopping the electrophoresis.
[0022] In one embodiment of the present invention, after obtaining the electrophoretic protein gel after electrophoresis in the electrophoresis tank, the method further includes: fixing the electrophoretic protein gel to obtain a fixed protein gel; and staining and decolorizing the fixed protein gel to obtain a decolorized protein gel.
[0023] In one embodiment of the present invention, the step of fixing the electrophoretic protein gel to obtain a fixed protein gel specifically includes: placing the electrophoretic protein gel in a fixation culture dish, and adding 0.2%-0.8% glutaraldehyde and 30% ethanol to the fixation culture dish for the fixation treatment to obtain a fixed protein gel; the step of staining and destaining the fixed protein gel to obtain the separation detection band specifically includes: placing the fixed protein gel in a staining culture dish and adding staining solution to the staining culture dish to obtain a stained protein gel; and adding destaining solution to the stained protein gel, microwaving it, and then placing it in a destaining shaker for destaining for 40-60 minutes to obtain a destaining protein gel.
[0024] As can be seen from the above, the technical features of the present invention can have one or more of the following beneficial effects:
[0025] The electrophoretic gel plate for detecting GLP-1 analog content of this invention has a three-layer structure: a stacking gel layer, a separator gel layer, and a separating gel layer. GLP-1 analogs in the sample are concentrated through the large-pore stacking gel layer and then enter the separator gel layer. In the separator gel layer, the GLP-1 analogs, with an acrylamide mixture concentration of 0.08-0.12 g / ml, exhibit moderate retention of GLP-1 analogs with a molecular weight of approximately 3-7 KD, while showing strong retention of substances greater than 10 KD and high permeability to substances below 3 KD. This allows the GLP-1 analogs entering the separating gel layer (0.15-0.17 g / ml acrylamide mixture) to separate from other substances, forming clear GLP-1 analog bands in the 3-7 KD region, thus meeting the requirements for GLP-1 analog detection.
[0026] Furthermore, the method for preparing an electrophoretic gel plate for detecting GLP-1 analog content provided by this invention controls the concentration of acrylamide mixture in the gel preparation solution to a specific value, thereby forming a gel electrophoresis plate with the pores required for detecting GLP-1 analogs. Simultaneously, the method for detecting GLP-1 analogs provided by this invention can complete the detection of GLP-1 analogs. Attached Figure Description
[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 This is a schematic diagram of the separated detection strips obtained in Embodiment 1 of this application.
[0029] Figure 2 This is a schematic diagram of the separated detection bands obtained in Comparative Example 1 of this application.
[0030] Figure 3 This is a schematic diagram of the separated detection bands obtained in Comparative Example 2 of this application. Detailed Implementation
[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0032] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such terms can be used interchangeably where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.
[0033] It should also be noted that the division of multiple embodiments in this invention is only for the convenience of description and should not constitute a special limitation. Features in various embodiments can be combined and referenced in each other without contradiction.
[0034] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features described herein can be combined with each other.
[0035] This invention provides an electrophoresis gel plate for detecting the content of GLP-1 analogs. For example, the electrophoresis gel plate includes, in sequence along the electrophoresis direction, a stacking gel layer, a separator gel layer, and a separating gel layer; wherein, the concentration of the acrylamide mixture in the separating gel layer is 0.16 g / ml, and the length of the separating gel layer along the electrophoresis direction is 6-9 cm; the concentration of the acrylamide mixture in the separator gel layer is 0.10 g / ml, and the length of the separator gel layer along the electrophoresis direction is 1.5-2.5 cm; the concentration of the acrylamide mixture in the stacking gel layer is 0.04 g / ml, and the length of the stacking gel layer along the electrophoresis direction is 2.5-4 cm.
[0036] Specifically, this invention also provides a method for preparing the above-mentioned electrophoresis gel plate, comprising the following steps:
[0037] S10: Mix acrylamide mixture, water, and gel buffer to form a separating gel solution, a separator gel solution, and a stacking gel solution for preparing electrophoresis gel plates;
[0038] S12: The separating gel preparation solution with an acrylamide mixture content of 0.16 g / ml is added to the gel preparation plate, and after solidification, a first transition gel preparation plate with a separating gel layer with a length of 6-9 cm along the electrophoresis direction is formed.
[0039] S14: The acrylamide mixture with a content of 0.10 g / ml is added to the first transition plate, and after solidification, a second transition plate with a separation layer having a length of 1.5-2.5 cm along the electrophoresis direction is formed.
[0040] S16: The concentrated gel preparation solution with an acrylamide mixture content of 0.04 g / ml is added to the second transition gel preparation plate, and after solidification, the electrophoretic gel plate with a concentrated gel layer having a length of 2.5 cm-4 cm along the electrophoresis direction is formed.
[0041] For example, the gel casting plate mentioned above is a specialized device for preparing electrophoretic gels. The gel buffer mentioned above includes, for example, tris(hydroxymethyl)aminomethane (Tris) and sodium dodecyl sulfate (SDS).
[0042] Further, before step S10, the acrylamide mixture is obtained by mixing acrylamide and methylenebisacrylamide at a mass ratio of 29:1.
[0043] The separating gel solution in step S10 also includes 5% glycerol.
[0044] In this invention, by adding 5% glycerol to the separating gel preparation solution, the spatial structure and dispersion conditions of the separating gel layer are changed, so that the GLP-1 analog can be stably dispersed in the separating gel layer environment when passing through the separating gel layer, and during electrophoresis, it is not only affected by the formation of pores in the gel, but also by the effect of glycerol in it.
[0045] In one specific implementation, step S12 specifically includes:
[0046] S122: Add ammonium persulfate to the separating gel preparation solution at a rate of 0.5 ug / ml, and add tetramethylethylenediamine to the separating gel preparation solution at a rate of 1 ug / ml to obtain the separating gel;
[0047] S124: The separating adhesive is added to the gel-making plate, and after solidification, it forms the first transition gel-making plate with a separating adhesive layer having a length of 6-9 cm along the electrophoresis direction.
[0048] In one specific implementation, step S14 specifically includes:
[0049] S142: Add ammonium persulfate to the separator adhesive solution at 0.83 ug / ml, and add tetramethylethylenediamine to the separator adhesive solution at 1.66 ug / ml to obtain the separator adhesive;
[0050] S144: The separator adhesive is added to the first transition adhesive plate, and after solidification, a second transition adhesive plate is formed with a separator adhesive layer having a length of 1.5-2.5 cm along the electrophoresis direction.
[0051] In one specific implementation, step S16 specifically includes:
[0052] S162: Add ammonium persulfate to the concentrated gel preparation solution at a concentration of 0.83 ug / ml, and add tetramethylethylenediamine to the concentrated gel preparation solution at a concentration of 1.66 ug / ml to obtain the concentrated gel;
[0053] S164: The concentrated gel is added to the second transition gel plate, and after solidification, the electrophoretic gel plate is formed with the concentrated gel layer having a length of 2.5cm-4cm along the electrophoresis direction.
[0054] It should be noted that the aforementioned ammonium persulfate is, for example, a coagulant, and tetramethylethylenediamine is, for example, a coagulant accelerator. Adding ammonium persulfate and tetramethylethylenediamine can cause the adhesive solution to solidify into a colloid. Specifically, the adhesive solution needs to be prepared first, and then ammonium persulfate and tetramethylethylenediamine are added just before being poured into the adhesive forming plate.
[0055] As described above, step S164 specifically includes: pouring the stacking gel into the second transition gel plate and pouring it to the top of the second transition gel plate; inserting the electrophoresis comb and placing it until the stacking gel solidifies; and removing the electrophoresis comb to obtain the electrophoresis gel plate with the sample loading port. The sample loading port, for example, is formed after removing the electrophoresis comb.
[0056] The present invention provides a method for preparing an electrophoretic gel plate for detecting GLP-1 analog content. By controlling the concentration of acrylamide mixture in the gel preparation solution to a specific value, a gel electrophoresis plate with the pores required for detecting GLP-1 analogs is formed.
[0057] On the other hand, embodiments of the present invention also provide, for example, a method for detecting GLP-1 analogs, which utilizes, for example, the electrophoretic gel plate mentioned above. Specifically, the method for detecting GLP-1 analogs includes:
[0058] S20: Add the sample to be tested to the aforementioned electrophoresis gel plate;
[0059] S22: The protein gel obtained after electrophoresis in an electrophoresis tank.
[0060] The protein gel after electrophoresis has separation detection bands.
[0061] Specifically, the electrophoresis tank mentioned in S22 includes an inner tank and an outer tank, and step S22 specifically includes:
[0062] S222: Install the electrophoresis gel plate into the inner tank of the electrophoresis tank, add a negative electrophoresis buffer containing 0.1 mol / L tris(hydroxymethyl)aminomethane to the inner tank, and add a positive electrophoresis buffer containing 0.2 mol / L tris(hydroxymethyl)aminomethane to the outer tank.
[0063] The positive electrode electrophoresis buffer contains tris(hydroxymethyl)aminomethane (Tris), and the negative electrode electrophoresis buffer contains tris(hydroxymethyl)aminomethane (Tris), tris(hydroxymethyl)glycine, and sodium dodecyl sulfate (SDS).
[0064] Furthermore, the electrophoresis gel plate includes a staining agent, such as bromophenol blue. Step S22 also includes:
[0065] S224: Connect the electrophoresis tank to a power source, and electrophore the electrophoresis gel plate for 10-15 minutes under an electric field strength of 3-5V / cm, and then electrophore it under an electric field strength of 8V / cm-15V / cm until the staining agent reaches the bottom of the electrophoresis gel plate, and then stop the electrophoresis.
[0066] Furthermore, the method for detecting GLP-1 analogs, after step S22, further includes:
[0067] S24: After immobilizing the electrophoretic protein gel, a fixed protein gel is obtained;
[0068] S26: After staining and decolorizing the fixed protein glue, a decolorized protein glue is obtained.
[0069] Specifically, step S24 includes: placing the electrophoretically deposited protein gel into a fixation culture dish, and adding 0.2%-0.8% glutaraldehyde and 30% ethanol to the fixation culture dish for fixation treatment to obtain the fixed protein gel.
[0070] Step S26 specifically includes:
[0071] S262: Place the fixed protein gel into a staining culture dish and add staining solution to the staining culture dish to obtain stained protein gel; and
[0072] S264: Add decolorizing solution to the stained protein gel, microwave it, and then place it in the decolorizing shaker for 40-60 minutes to obtain the decolorized protein gel.
[0073] The staining solution includes Coomassie Brilliant Blue R250, and the destaining solution includes ethanol and acetic acid. The protein gel after electrophoresis is fixed, stained, and destained to make the separation and detection bands clearer.
[0074] The method for detecting GLP-1 analogs provided by this invention can achieve accurate detection of GLP-1 analogs.
[0075] The following section provides specific examples illustrating the preparation method of electrophoretic gel plates for detecting GLP-1 analog content and the method for detecting GLP-1 analogs, based on the steps described above.
[0076] First, the electrophoresis gel plates were prepared. Specifically, an acrylamide mixture, water, gel buffer, and glycerol were mixed to obtain separating gel, separator gel, and stacking gel solutions, respectively. The separating gel solution contained 0.16 g / ml of acrylamide mixture and 5% glycerol. Following this, the separator gel solution contained 0.10 g / ml of acrylamide mixture, and the stacking gel solution contained 0.04 g / ml of acrylamide mixture. Ammonium persulfate at concentrations of 0.5 μg / ml, 0.83 μg / ml, and 0.83 μg / ml, and tetramethylethylenediamine at concentrations of 1 μg / ml, 1.66 μg / ml, and 1.66 μg / ml, respectively, were added to the separating gel, separator gel, and stacking gel solutions to obtain the separating gel, separator gel, and stacking gel, respectively. A separating gel is added to the gel casting plate, and after solidification, a first transition gel casting plate with a separating gel layer of 6-9 cm in length along the electrophoresis direction is formed. A separator gel is added to the first transition gel casting plate, and after solidification, a second transition gel casting plate with a separator gel layer of 1.5-2.5 cm in length along the electrophoresis direction is formed. A stacking gel is added to the second transition gel casting plate and poured to the top of the second transition gel casting plate. An electrophoresis comb is inserted and placed until the stacking gel solidifies. After removing the electrophoresis comb, an electrophoresis gel plate with a sample loading port is obtained, wherein the electrophoresis gel plate has a stacking gel layer of 2.5 cm-4 cm in length along the electrophoresis direction. This completes the preparation of the electrophoresis gel plate.
[0077] It should be noted that the acrylamide mixture is, for example, obtained by mixing acrylamide and methylenebisacrylamide, with a mass ratio of acrylamide to methylenebisacrylamide of 29:1. The gel buffer is, for example, obtained by dissolving tris(hydroxymethyl)aminomethane and sodium dodecyl sulfate in a solvent, such as water and hydrochloric acid. Electrophoresis using the electrophoresis gel plate provided in this application can yield clear separation and detection bands.
[0078] GLP-1 analogues were then detected using the aforementioned electrophoresis gel plates.
[0079] First, before detecting GLP-1 analogs, the sample needs to be pretreated. Pretreatment involves adding glycerol to the supernatant of the sample, then adding loading buffer and mixing thoroughly. The sample is then boiled in a metal bath at 100°C for 10 minutes for denaturation. The loading buffer may contain, for example, sodium dodecyl sulfate (SDS), a reducing agent (e.g., β-mercaptoethanol), and bromophenol blue (staining agent). The sample is then added sequentially to the electrophoresis gel plate through the sample loading port.
[0080] Next, after electrophoresis in the electrophoresis tank, a post-electrophoretic protein gel is obtained, which contains separation detection bands. Specifically, the electrophoresis gel plate is first installed in the inner tank of the electrophoresis tank, and negative electrophoresis buffer containing 0.1 mol / L tris(hydroxymethyl)aminomethane is added to the inner tank to the maximum level. Positive electrophoresis buffer containing 0.2 mol / L tris(hydroxymethyl)aminomethane is added to the outer tank of the electrophoresis tank to 4 / 5 of its capacity. The negative electrophoresis buffer includes tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)glycine, and sodium dodecyl sulfate, while the cation electrophoresis buffer includes tris(hydroxymethyl)aminomethane. Following this, the electrophoresis tank is connected to a power source, and the electrophoresis gel plate is electrophoresed for 10-15 minutes at an electric field strength of 3-5 V / cm. Then, electrophoresis is stopped at an electric field strength of 8 V / cm-15 V / cm until the staining agent (bromophenol blue) reaches the bottom of the electrophoresis gel plate, thus obtaining the post-electrophoretic protein gel.
[0081] In addition, the electrophoretic protein gel is sequentially fixed, stained, and destained to obtain a destaining protein gel with clearer separation bands. Specifically, the electrophoretic protein gel is first placed in a fixation culture dish, and 0.2%-0.8% glutaraldehyde and 30% ethanol are added to the fixation culture dish for fixation, resulting in a fixed protein gel. Fixing the electrophoretic protein gel prevents protein detachment during the subsequent destaining process. Following this, the fixed protein gel is placed in a staining culture dish, and staining solution is added to the staining culture dish to obtain a stained protein gel. Then, destaining solution is added to the stained protein gel, and after microwave heating, it is placed in a destaining shaker for 40-60 minutes to obtain a destaining protein gel. The staining solution mentioned above includes Coomassie Brilliant Blue R250, and the destaining solution mentioned above includes ethanol and acetic acid.
[0082] Finally, the separated detection bands were photographed for analysis.
[0083] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.
[0084] Example 1:
[0085] 1.1 Sample Pretreatment
[0086] Add 10% glycerol to the supernatant of the sample to be tested, mix with 5X (5x concentrated solution) loading buffer, and boil in a metal bath at 100°C for 10 min to denature.
[0087] 1.2 Reagent Preparation
[0088]
[0089]
[0090] 1.3 Preparation of electrophoresis gel plates
[0091] After installing the gel casting plate according to the instructions for the gel casting machine and confirming there are no leaks, prepare an electrophoresis gel plate containing electrophoretic gels (separating gel, diaphragm gel, and stacking gel). The components of the separating gel, diaphragm gel, and stacking gel are shown in Table 1.
[0092] Table 1. Formulation of separating gel, separating gel, and stacking gel.
[0093]
[0094] 1) Selection of glue-making board: Select a glue-making board with a thickness of 1.5mm * 12cm (length).
[0095] 2) Preparation of separating gel: Prepare 15ml of separating gel according to the preparation method in Table 1.
[0096] 3) Injecting the separating adhesive: Inject the prepared separating adhesive into about 3 / 5 of the glue-making plate, injecting 6ml into each glue-making plate;
[0097] 4) Water seal: Add about 1.5 ml of deionized water to the gel casting plate containing the separating gel using a 200 μl gun, let it stand at room temperature for about 45 minutes, and pour off the deionized water after the gel solidifies to obtain the first transition gel casting plate with a separating gel layer of 6-9 cm in length along the electrophoresis direction.
[0098] 5) Injecting the interlayer adhesive: Prepare the interlayer adhesive according to the preparation method in Table 1. Add 1.5ml of interlayer adhesive to each adhesive plate, and prepare a total of 5ml of interlayer adhesive for two plates. Inject the interlayer adhesive into the first transition adhesive plate.
[0099] 6) Water seal: Add about 1.5 ml of deionized water to the gel casting plate containing the separator gel using a 200 μl gun, let it stand at room temperature for about 30 minutes, and pour off the deionized water after the separator gel solidifies to obtain a second transition gel casting plate with a separator gel layer with a length of 1.5-2.5 cm along the electrophoresis direction.
[0100] 7) Prepare the stacking gel: Prepare the stacking gel according to the preparation method in Table 1, and prepare a total of 5ml.
[0101] 8) Pouring concentrated adhesive: Pour the prepared concentrated adhesive into the second transition glue-making plate, up to the top of the glue-making plate;
[0102] 9) Insert the comb: Insert the electrophoresis comb with the numbers on the comb facing inwards, and let it stand for 30 minutes until the stacking gel solidifies to obtain an electrophoresis gel plate with a stacking gel layer of 2.5cm-4cm in length along the electrophoresis direction. The electrophoresis gel plate has a sample loading port near the stacking gel layer.
[0103] 1.4 Detection of GLP-1 analogs
[0104] 1) Add electrophoresis buffer: Install the electrophoresis tank according to the instructions. Install the prepared electrophoresis gel plate into the inner tank of the electrophoresis tank, add negative electrophoresis buffer (1x concentrate) to the inner tank to the highest point, and add positive electrophoresis buffer (1x concentrate) to the outer tank to 4 / 5 of the tank.
[0105] 2) Spotting: After removing the comb, add the test sample, control sample and protein label in sequence. The sample contains a GLP-1 analog with greater than 95% homology to human GLP-1. The GLP-1 analog has fatty acid chains added due to modification.
[0106] 3) Electrophoresis: Use the DYY-6C electrophoresis apparatus power supply from Beijing Liuyi Biotechnology Co., Ltd. as the power source. First, perform electrophoresis at a constant voltage of 30V for 15 minutes, then at 100V for 60 minutes, and finally switch to 140V to the lowest point to complete the electrophoresis.
[0107] 4) Gel cutting: Use a gel cutting tool to pry open the electrophoresis gel plate, remove the protein gel after electrophoresis, and place it in a fixation culture dish;
[0108] 5) Fixation: After electrophoresis, place the protein gel into a fixation culture dish, add an appropriate amount of fixative, and then place it in a decolorizing shaker for 45 minutes to obtain the fixed protein gel.
[0109] 6) Staining: Place the fixed protein gel into a staining culture dish, add an appropriate amount of decolorizing solution, heat in a microwave oven for 1 minute, and then stain in a decolorizing shaker for 70 minutes to obtain the stained protein gel.
[0110] 7) Decolorization: After recovering the staining solution, add an appropriate amount of decolorizing solution, heat in a microwave oven for 1 minute, stain in a decolorizing shaker for 50 minutes, repeat several times until the bands are clear, to obtain the decolorized protein gel;
[0111] 8) Take photos: Take photos to analyze the strips.
[0112] The bands obtained after electrophoresis are as follows: Figure 1 As shown, from Figure 1 As can be clearly seen in the image, the 6.5kD GLP-1 analog band in the sample is very clear and well distinguishable from the other bands.
[0113] Comparative Example 1:
[0114] Under the premise that other implementation conditions are basically the same as those in Example 1, the electrophoretic gel plate of Comparative Example 1 was prepared using the electrophoretic gel scheme in Table 2.
[0115] Table 2. Composition of Electrophoresis Gel Formulation
[0116]
[0117]
[0118] The bands obtained after electrophoresis according to Comparative Example 1 are as follows: Figure 2 As shown, although bands of GLP-1 analogs were formed in the region below 10kD, these bands could not be clearly distinguished from bands of other molecular weights. That is, the bands formed by substances with molecular weights less than 10kD and GLP-1 analogs overlapped, making it impossible to determine whether GLP-1 analogs existed or to calculate the content of GLP-1 analogs.
[0119] Comparative Example 2:
[0120] Under the premise that other implementation conditions are basically the same as those in Example 1, the electrophoretic gel plate of Comparative Example 1 was prepared using the electrophoretic gel scheme in Table 3.
[0121] Table 3. Composition of Electrophoresis Gel Formulation
[0122]
[0123] The bands obtained after electrophoresis according to Comparative Example 2 are as follows: Figure 3 As shown, no GLP-1 analog bands were formed in the region below 10 kD, making it impossible to determine the presence and specific content of GLP-1 analogs.
[0124] Based on the above information, it is clear that only the electrophoresis gel plate for detecting GLP-1 analog content and the method for separating and detecting GLP-1 analogs provided in this application can meet the requirements for the separation and detection of GLP-1 analogs, thereby solving the problem that the existing SDS-PAGE protein electrophoresis has poor separation effect on GLP-1 analog proteins and is difficult to accurately detect GLP-1 analogs.
[0125] It is understood that the foregoing embodiments are merely illustrative examples of the present invention. Provided that the technical features do not conflict, the structure is not contradictory, and the purpose of the invention is not violated, the technical solutions of the various embodiments can be arbitrarily combined and used.
[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. An electrophoretic gel plate for detecting the content of GLP-1 analogues, characterized in that, The electrophoresis gel plate comprises, in sequence along the electrophoresis direction, a stacking gel layer, a separator gel layer, and a separating gel layer. The concentration of the acrylamide mixture in the separating gel layer is 0.15-0.17 g / ml, and the length of the separating gel layer along the electrophoresis direction is greater than 5 cm. The concentration of the acrylamide mixture in the separator gel layer is 0.08-0.12 g / ml, and the length of the separator gel layer along the electrophoresis direction is 1-3 cm. The concentration of the acrylamide mixture in the stacking gel layer is 0.02-0.05 g / ml, and the length of the stacking gel layer along the electrophoresis direction is greater than 1 cm.
2. A method for preparing an electrophoretic gel plate for detecting the content of GLP-1 analogs, characterized in that, Includes the following steps: Acrylamide mixture, water, and gel buffer are mixed to form a separating gel preparation solution, a separator gel preparation solution, and a stacking gel preparation solution for preparing electrophoresis gel plates; the separating gel preparation solution with an acrylamide mixture content of 0.15-0.17 g / ml is added to the gel plate, and after solidification, a first transition gel plate with a separating gel layer having a length greater than 5 cm along the electrophoresis direction is formed. The acrylamide mixture with a content of 0.08-0.12 g / ml is added to the first transition plate, and after solidification, a second transition plate with a separation layer with a length of 1-3 cm along the electrophoresis direction is formed. The concentrated gel solution with an acrylamide mixture content of 0.02-0.05 g / ml is added to the second transition gel plate. After solidification, the electrophoresis gel plate is formed with a concentrated gel layer having a length greater than 1 cm along the electrophoresis direction. The concentrated gel layer of the electrophoresis gel plate has a sample loading port.
3. The method for preparing an electrophoretic gel plate for detecting GLP-1 analog content as described in claim 2, characterized in that, include: The acrylamide mixture is obtained by mixing acrylamide and methylenebisacrylamide at a mass ratio of (20-29):1, wherein the separating gel solution contains 4-6% glycerol.
4. The method for preparing an electrophoretic gel plate for detecting GLP-1 analog content as described in claim 2, characterized in that, The step of adding the separating gel preparation solution with an acrylamide mixture content of 0.15-0.17 g / ml to the gel preparation plate, and forming a first transition gel preparation plate with a separating gel layer having a length greater than 5 cm along the electrophoresis direction after solidification, includes: Ammonium persulfate was added to the separating gel preparation solution at a concentration of 0.4-0.6 ug / ml, and tetramethylethylenediamine was added to the separating gel preparation solution at a concentration of 0.8-1.2 ug / ml to obtain the separating gel. The separating gel is added to the gel-making plate, and after solidification, a first transition gel-making plate with a separating gel layer having a length of 6-9 cm along the electrophoresis direction is formed. The step of adding the acrylamide mixture with a content of 0.08-0.12 g / ml to the first transition plate, and forming a second transition plate with a separating adhesive layer having a length of 1-3 cm along the electrophoresis direction after solidification, includes: Ammonium persulfate was added to the separator adhesive solution at a concentration of 0.7-0.9 ug / ml, and tetramethylethylenediamine was added to the separator adhesive solution at a concentration of 1.5-1.7 ug / ml to obtain the separator adhesive. The separator adhesive is added to the first transition adhesive plate, and after solidification, a second transition adhesive plate is formed with a separator adhesive layer having a length of 1.5-2.5 cm along the electrophoresis direction. The step of adding the concentrated gel solution with an acrylamide mixture content of 0.02-0.05 g / ml to the second transition gel plate, and forming the electrophoretic gel plate after solidification, comprising: Ammonium persulfate was added to the concentrated gel preparation solution at a concentration of 0.7-0.9 ug / ml, and tetramethylethylenediamine was added to the concentrated gel preparation solution at a concentration of 1.5-1.7 ug / ml to obtain the concentrated gel. The concentrated gel is added to the second transition gel plate, and after solidification, an electrophoretic gel plate is formed with a concentrated gel layer having a length of 2.5cm-4cm along the electrophoresis direction.
5. The method for preparing an electrophoretic gel plate for detecting GLP-1 analog content as described in claim 4, characterized in that, The step of adding the stacking gel to the second transition gel plate, and forming an electrophoretic gel plate with a stacking gel layer having a length of 2.5cm-4cm along the electrophoresis direction after solidification, includes: The concentrated gel is poured into the second transition gel plate and poured to the top of the second transition gel plate. An electrophoresis comb is inserted and placed until the concentrated gel solidifies. After removing the electrophoresis comb, the electrophoresis gel plate with the sample dispensing port is obtained.
6. A method for detecting GLP-1 analogs, characterized in that, include: The sample to be tested is added to the electrophoresis gel plate prepared by the method of preparing the electrophoresis gel plate for detecting the content of GLP-1 analogs according to any one of claims 1 or 2-5, and electrophoresis is performed in an electrophoresis tank to obtain the post-electrophoresis protein gel; wherein, the post-electrophoresis protein gel has a separation detection band, and the molecular weight of the GLP-1 analog is 3KD-7KD.
7. The method for detecting GLP-1 analogs as described in claim 6, characterized in that, The process of obtaining a post-electrophoretic protein gel after electrophoresis in an electrophoresis tank includes: The electrophoresis gel plate is installed in the inner tank of the electrophoresis tank, and a negative electrophoresis buffer containing 0.05-0.15 mol / L tris(hydroxymethyl)aminomethane is added to the inner tank, and a positive electrophoresis buffer containing 0.15-0.25 mol / L tris(hydroxymethyl)aminomethane is added to the outer tank of the electrophoresis tank.
8. The method for detecting GLP-1 analogs as described in claim 6, characterized in that, The electrophoretic gel plate contains a staining agent; The process of obtaining an electrophoretic protein gel after electrophoresis in an electrophoresis tank further includes: Connect the electrophoresis tank to a power source, and electrophore the electrophoresis gel plate for 10-15 minutes under an electric field strength of 3-5V / cm, and then electrophore it under an electric field strength of 8V / cm-15V / cm until the staining agent reaches the bottom of the electrophoresis gel plate, and then stop the electrophoresis.
9. The method for detecting GLP-1 analogs as described in any one of claims 6-8, characterized in that, After obtaining the electrophoretic protein gel after electrophoresis in the electrophoresis tank, the method further includes: After immobilizing the electrophoretic protein gel, a fixed protein gel is obtained. After staining and decolorizing the fixed protein glue, decolorized protein glue is obtained.
10. The method for detecting GLP-1 analogs as described in claim 9, characterized in that, The process of fixing the electrophoretically deposited protein gel to obtain the fixed protein gel specifically includes: The electrophoresed protein gel was placed in a fixation culture dish, and 0.2%-0.8% glutaraldehyde and 30% ethanol were added to the fixation culture dish for fixation treatment to obtain the fixed protein gel. The process of staining and destaining the fixed protein gel to obtain the separated detection bands specifically includes: The fixed protein gel was placed in a staining culture dish, and staining solution was added to the staining culture dish to obtain the stained protein gel; and Add a decolorizing solution to the stained protein gel, microwave it, and then place it in the decolorizing shaker for 40-60 minutes to obtain the decolorized protein gel.