Incubation box for incubating protein immunoblotting antibody
By designing a protein immunoimprint antibody incubation box, using a support plate and a hydrophobic isolation baffle combined with digital temperature and humidity control, the problem of multi-protein detection in protein immunoimprint experiments was solved, and efficient and accurate antibody incubation effect was achieved.
Patent Information
- Application Number
- CN202422145951.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-02
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2034-09-02
AI Technical Summary
In existing protein immunoimprint experiments, multiple protein molecules cannot be detected simultaneously, and there are problems such as high economic costs, long experiment time, inaccurate results and poor repeatability during the antibody incubation process.
An incubation box for protein immunoimprint antibody incubation is designed, including a jacket box and a membrane-carrying box, a support plate and a hydrophobic isolation baffle, combined with a digital constant temperature and humidity controller, multi-region antibody incubation is realized, and the temperature and humidity sensor and timing module are used to ensure accurate control of experimental conditions.
It realizes the detection of multiple protein molecules simultaneously without damaging the membrane, shortens the experimental time, improves detection efficiency and accuracy, reduces the amount of antibody used, and ensures the repeatability and economic benefits of the experiment.
Smart Images
Figure CN223091965U_ABST
Abstract
Description
Technical Field
[0001] The utility model belongs to the technical field of protein expression level detection and antibody incubation, and particularly relates to an incubation box for antibody incubation in protein immunoblotting. Background Art
[0002] Protein immunoblotting experiment, namely Western blotting, is a common experimental method applied in molecular biology, biochemistry and immunology. It mainly includes five parts: protein sample preparation, SDS-PAGE gel electrophoresis, protein transfer, antibody incubation and imaging analysis.
[0003] In protein immunoblotting experiments, it is often necessary to detect the expression levels of at least two or more specific protein molecules. However, in the process of antibody incubation, the currently commonly used methods are as follows:
[0004] 1. Incubate the whole membrane carrying proteins with specific antibodies. Its disadvantage is that the price of specific polyclonal antibodies or monoclonal antibodies is high. The price of 10 μL of specific antibodies is often as high as thousands of yuan. A large amount of antibodies is required to incubate the whole membrane, resulting in high economic costs and only one specific protein can be detected at a time, with low detection efficiency. If multiple proteins need to be detected in one experiment, only after each protein is detected, the whole membrane is immersed in an antibody stripping solution with a specific pH environment for elution and then re-incubated. This method will increase the experimental time and may have unknown effects on the proteins adsorbed on the membrane due to the specific pH environment of the stripping solution, resulting in inaccurate experimental data.
[0005] 2. According to the indication of the Marker of standard pre-stained protein molecular weight, the membrane carrying proteins is cut and divided according to the molecular weight of a certain protein molecule to be detected to detect multiple proteins. Its disadvantage is that it causes irreparable damage to the membrane, and if the molecular weight of the protein molecule to be detected changes slightly due to unknown reasons such as post-translational modification, it may be impossible to detect the positive result due to cutting the membrane and there is no remedy.
[0006] At the same time, the current antibody incubation conditions generally use a 4°C refrigerator to incubate overnight for 12 - 16 hours. However, as a relatively large environmental space, the refrigerator cannot accurately control the precise temperature and humidity in each area, which will cause large experimental errors in different batches of experiments, unable to ensure the repeatability between different batches of experiments, and cause great interference to the experimental results. Content of the Utility Model
[0007] The purpose of the present utility model is to provide an incubation box for protein immunoblot antibody incubation, which solves the problems that in the existing experimental scheme during antibody incubation, it is impossible to detect the expression of multiple proteins simultaneously, resulting in an increase in experimental time and inaccuracy, or when detecting multiple proteins simultaneously, the membrane needs to be cut, causing irreparable damage.
[0008] The present utility model is realized through the following technical solutions:
[0009] An incubation box for protein immunoblot antibody incubation, comprising an outer box and a membrane-carrying box. The membrane-carrying box is arranged in the outer box to form a double-layer box structure;
[0010] There are multiple support plates in the outer box. The membrane-carrying box is placed on the support plates, and the support plates are used to hold up the membrane-carrying box;
[0011] The bottom plate of the membrane-carrying box serves as the working substrate. There are multiple clamping members in the membrane-carrying box, and each clamping member is used to place a hydrophobic isolation baffle;
[0012] During antibody incubation, hydrophobic isolation baffles are pressed on the detection film on the working substrate as needed to divide the detection film into multiple detection areas;
[0013] A moisturizing medium is filled in the area below the working substrate.
[0014] Furthermore, the shell of the membrane-carrying box is coated with a heat-insulating layer.
[0015] Furthermore, there are multiple water-permeable holes on the support plates.
[0016] Furthermore, the width of the membrane-carrying box is smaller than that of the outer box.
[0017] Furthermore, both the working substrate and the hydrophobic isolation baffle are made of hydrophobic materials.
[0018] Furthermore, both the outer box and the membrane-carrying box are made of plastic materials.
[0019] Furthermore, the working substrate is a corrugated plate.
[0020] Furthermore, the hydrophobic isolation baffle is placed directly above the support plate.
[0021] Furthermore, a digital constant-temperature temperature controller and a digital humidity controller are provided in the membrane-carrying box;
[0022] A display screen is correspondingly arranged on the outer box. The display screen is electrically connected to the digital constant-temperature temperature controller and the digital humidity controller, and is used to display the humidity and temperature inside the membrane-carrying box.
[0023] Furthermore, an ultrasonic humidifier, a heater, and a thermoelectric cooler are provided in the outer box. The ultrasonic humidifier, the heater, and the thermoelectric cooler are all equipped with fans.
[0024] Further, a timing module is provided on the outer box.
[0025] Further, an Internet of Things two-dimensional code is provided on the outer box.
[0026] Compared with the prior art, the utility model has the following beneficial technical effects:
[0027] The utility model discloses an incubation box for protein immunoblot antibody incubation, which includes an outer box and a membrane-carrying box. The membrane-carrying box is arranged in the outer box. There are multiple support plates in the outer box, and the membrane-carrying box is placed on the support plates. A hydrophobic isolation baffle is arranged in the membrane-carrying box. Through the combination of the membrane-carrying box and the hydrophobic isolation baffle, the whole membrane carrying proteins can be non-destructively separated at the required specific positions. On the premise of reducing the use of antibodies, irreversible damage to the membrane can be avoided, and the purpose of simultaneously detecting multiple specific protein molecules can be achieved; on the basis of shortening the experimental period, both economic benefits are improved, and the working efficiency and accuracy of protein detection are also improved.
[0028] Further, multiple water through holes are provided on the support plates to ensure the fluidity of distilled water and better moisturize the PVDF membrane.
[0029] Further, the width of the membrane-carrying box is smaller than the width of the outer box, so that the water vapor below can flow to the upper part, accelerating the flow of water vapor.
[0030] Further, the working substrate is used to carry the PVDF membrane that needs to be incubated with antibodies. The working substrate is designed as a corrugated board, which can reduce the mutual flow and crosstalk of the antibody liquids added between different separated areas.
[0031] Further, this incubation box also integrates a temperature and humidity sensor, a temperature control module, a humidification module, a timing module and an Internet of Things two-dimensional code. The temperature and humidity of the intelligent incubation box can be adjusted in time and accurately timed, so as to ensure the repeatability and accuracy between different batches of experiments. At the same time, the high humidity environment inside the incubation box can reduce evaporation, thereby reducing the usage amount of specific antibodies and improving economic benefits. At the same time, it can be interconnected with the mobile phone by identifying the two-dimensional code. During the overnight incubation of antibodies for 12-16 hours, the temperature and humidity conditions of the incubation process can be remotely and dynamically monitored, and the experimental conditions can be recorded. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 It is a schematic diagram of the overall structure of an incubation box for protein immunoblot antibody incubation of the utility model;
[0033] Figure 2Schematic diagram of the assembly of the membrane carrier box and the outer box in an incubation box for protein immunoblot antibody incubation of the present utility model;
[0034] Figure 3 Schematic diagram of the assembly of the membrane carrier box without the clamping member and the outer box;
[0035] Figure 4 For Figure 1 Top view;
[0036] Figure 5 Schematic diagram of the membrane carrier box;
[0037] Figure 6 Schematic diagram of the clamping member;
[0038] Figure 7 Schematic diagram of the temperature control module and the humidification module;
[0039] Wherein, 1. Power supply; 2. Digital humidity controller; 3. Display screen; 4. Digital constant temperature controller; 5. Ultrasonic humidifier; 6. Heater; 7. Electronic refrigeration sheet; 8. Hydrophobic isolation baffle; 9. Clamping member; 10. Outer box; 11. Water level line; 12. Box cover; 13. Membrane carrier box; 14. Support plate; 15. Working substrate. Detailed implementation manners
[0040] In order to make the purpose, technical solutions and advantages of the present utility model clearer, the following further detailed description is given in conjunction with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present utility model, and are not used to limit the present utility model, that is, the described embodiments are only a part of the embodiments of the present utility model, rather than all embodiments.
[0041] The components described and shown in the drawings and embodiments of the present utility model can be arranged and designed in various different configurations. Therefore, the detailed description of the embodiments of the present utility model provided in the following drawings is not intended to limit the scope of the present utility model to be protected, but only represents a selected embodiment of the present utility model. Based on the drawings and embodiments of the present utility model, all other embodiments obtained by those skilled in the art without creative efforts belong to the protection scope of the present utility model.
[0042] It should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, element, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or also includes elements inherent to the process, element, method, article or device.
[0043] The main steps of the protein immunoblot experiment are as follows:
[0044] Protein sample preparation: After extracting the protein mixture from plant or animal tissues or cells, the quaternary structure of the protein is disrupted by a strong reducing agent such as β-mercaptoethanol, and the depolymerized protein molecules are fully combined with SDS to form a negatively charged protein-SDS complex.
[0045] SDS-PAGE gel electrophoresis: Under the action of an electric field, the protein-SDS complex migrates in the SDS-PAGE gel, and its migration speed is linearly related to the molecular weight of the protein. Eventually, the protein mixture shows a linear distribution in the gel according to the molecular weight, and can be calibrated according to the Marker of the standard pre-stained protein molecular weight, so as to judge the molecular weight of the protein at different positions.
[0046] Protein transfer and milk blocking: For convenient subsequent detection, the proteins separated by SDS-PAGE gel are transferred to a solid support under the action of an electric field. Commonly used solid supports include PVDF membranes, nitrocellulose membranes, nylon membranes, etc. In most current experiments, PVDF membranes are used. The unreacted sites on the solid support are blocked with bovine serum albumin to inhibit non-specific adsorption in subsequent experiments, and then antibody incubation can be carried out.
[0047] Antibody incubation and imaging analysis: The proteins adsorbed on the solid support can specifically recognize and bind to specific polyclonal antibodies or monoclonal antibodies through immune reactions. Finally, the intensity of the antibody specifically bound to a certain protein molecule is detected by chemiluminescence, color development or fluorescence, etc., so as to reflect the expression level of a certain protein molecule on the solid support.
[0048] The features and performance of the present utility model are further described in detail below in conjunction with the embodiments.
[0049] As Figures 1 - 3 shown, the present utility model discloses an incubation box for protein immunoblot antibody incubation, which includes an outer box 10 and a membrane-carrying box 13. The membrane-carrying box 13 is arranged in the outer box 10 to form a double-layer box structure.
[0050] More preferably, a box cover 12 is provided on the outer box 10, which can play a role in dust prevention and moisture retention for the membrane-carrying box 13.
[0051] More preferably, a heat-insulating layer is coated on the front, rear, left and right regions of the membrane-carrying box 13 to ensure the stability of the temperature inside the incubation box.
[0052] More preferably, the bottom of the membrane-carrying box 13 does not contact the bottom of the outer box 10. Therefore, a plurality of support plates 14 are provided on the bottom plate of the outer box 10 for supporting and placing the membrane-carrying box 13 in the outer box 10.
[0053] Correspondingly, as Figure 5 and Figure 6 shown, a plurality of clamping members 9 are provided on the side wall of the film carrier box 13. Two clamping members 9 serve as a set of clamping mechanisms, and the hydrophobic isolation baffle 8 can be flexibly pressed into the clamping member 9. The clamping member 9 is correspondingly arranged above the working substrate 15.
[0054] Specifically, as Figure 6 shown, each clamping member 9 includes two clamping plates, and the two clamping plates form a clamping groove. The hydrophobic isolation baffle 8 is embedded in the clamping groove and can be flexibly installed and disassembled.
[0055] As Figure 5 shown, the bottom plate of the film carrier box 13 serves as the working substrate 15. The working substrate 15 is designed as a corrugated plate and is used to carry the PVDF membrane that needs to incubate antibodies. The purpose of being designed as an arc is to reduce the mutual flow and crosstalk of the antibody liquids added between different partition regions.
[0056] Distilled water is filled in the area below the working substrate 15 to play a moisturizing role. Generally, the water level line 11 of the distilled water is at half of the height position of the upper support plate 14.
[0057] More preferably, in order to better moisturize the PVDF membrane, a plurality of water through holes are provided on the support plate 14 to ensure the fluidity of the distilled water.
[0058] As Figure 2 shown, the support plate 14 divides the inside of the box into multiple regions. The working substrate 15 is provided at the top of the support plate 14, and the PVDF membrane after protein transfer is placed on the working substrate 15; during antibody incubation, a plurality of hydrophobic isolation baffles 8 are arranged on the working substrate 15 according to needs to divide the PVDF membrane into multiple detection regions, without the need to cut the PVDF membrane, and multiple proteins can be detected at one time without mixing the primary antibodies.
[0059] As Figure 2 shown, for example, if a certain experiment needs to detect 2 specific protein molecules, only a small area of the PVDF membrane is needed. It can be pressed along the direction perpendicular to the detection strip of the PVDF membrane to form area A and area B, and antibodies are dropped in area A and area B respectively.
[0060] As Figure 4As shown, if it is necessary to detect Protein A with a molecular weight of 110 KD and Protein B with a molecular weight of 36 KD in a certain sample, by traditional detection means, it is necessary to use scissors to cut between 110 KD and 36 KD. This results in irreversible damage to the PVDF membrane, which will affect the subsequent detection of the expression levels of other proteins between 110 KD and 36 KD. Or, without cutting the PVDF membrane, detecting the protein molecules of 110 KD and 36 KD in batches will lead to a decrease in experimental efficiency and an increase in the amount of antibody used, resulting in a waste of time and reagents.
[0061] As Figure 4 shown, in the present utility model, the entire PVDF membrane (the gray area in the figure) is placed on the working substrate 15, and a hydrophobic isolation baffle 8 is placed at a certain position in the middle between the Protein A band and the Protein B band to separate the A area and the B area.
[0062] More preferably, in the design, the width of the membrane carrier box 13 is smaller than the width of the outer casing box 10, so that the water vapor below can flow to the upper part.
[0063] The hydrophobic isolation baffle 8 and the working substrate 15 are made of hydrophobic materials, such as made of polyolefin and paraffin, and have hydrophobicity.
[0064] In order to better detect the temperature and humidity environment in the membrane carrier box 13, a digital constant temperature controller 4 and a digital humidity controller 2 are provided in the membrane carrier box 13; a display screen 3 is correspondingly arranged on the outer casing box 10, and the display screen 3 is electrically connected to the digital constant temperature controller 4 and the digital humidity controller 2 for displaying the humidity and temperature in the membrane carrier box 13. The display screen 3, the digital constant temperature controller 4 and the digital humidity controller 2 are all connected to the power supply 1.
[0065] Furthermore, when the temperature and humidity do not meet the requirements, adjustment is needed through control means. In the present utility model, an ultrasonic humidifier 5, a heater 6 and a thermoelectric cooler 7 are added to the inner wall of the outer casing box 10 ， Just stagger from the area filled with distilled water.
[0066] The ultrasonic humidifier 5, the heater 6 and the thermoelectric cooler 7 are all equipped with fans to strengthen air circulation and promote humidification, heating and refrigeration.
[0067] The power supply 1 adopts an AC / DC power module to supply power to the entire system.
[0068] As Figure 7 shown, the positive and negative terminals of the power supply 1 of the digital constant temperature controller 4 and the digital humidity controller 2 are respectively connected to the positive and negative outputs of the power supply 1.
[0069] One of the output control terminals on the digital constant temperature controller 4 is connected to the positive pole of the power supply 1, and the other terminal serves as the humidification output terminal.
[0070] The output control terminals on the digital constant temperature controller 4 include a heating control terminal and a refrigeration control terminal. One of the terminals in the heating control terminal is connected to the positive pole of the power supply 1, and the other terminal serves as the heating output terminal; one of the terminals in the refrigeration control terminal is connected to the positive pole of the power supply 1, and the other terminal serves as the refrigeration output terminal.
[0071] The negative terminals of the heater 6, the electronic refrigeration sheet 7, and the ultrasonic humidifier 5 are respectively connected to the negative pole of the power supply 1. The positive pole of the heater 6 is respectively connected to the heating output terminal of the temperature controller, the positive pole of the refrigeration sheet is connected to the refrigeration output terminal of the temperature controller, and the ultrasonic humidifier 5 is connected to the humidification output terminal of the humidity controller.
[0072] The output control terminals of the temperature controller and the humidity controller are relay outputs.
[0073] Since the temperature requirement for antibody incubation is generally 2°C - 8°C. Specifically, the heating temperature of the temperature controller is set to start at 2°C and stop at 4°C; the refrigeration temperature is set to start at 8°C and stop at 6°C. This ensures that the temperature is within a reasonable range.
[0074] Since the humidity requirement for antibody incubation is generally 92% - 96%, the humidity is set to start when it is <92% and stop when it reaches 96%.
[0075] The process of temperature control is specifically as follows:
[0076] Plug in the power supply 1, turn on the power supply 1 switch K to supply power to the system.
[0077] When the temperature in the film carrier box 13 drops to the set temperature of 2°C, the heating relay closes. The positive pole of the heater 6 is connected to the positive pole of the power supply 1 through the heating relay, and the heater 6 is powered on to start heating. When the temperature reaches the stop temperature, the heating relay releases and stops heating.
[0078] When the temperature in the film carrier box 13 is higher than the set temperature of 8°C, the refrigeration relay closes. The positive pole of the electronic refrigeration sheet 7 is connected to the positive pole of the power supply 1 through the refrigeration relay, and the electronic refrigeration sheet 7 starts to be powered on for refrigeration. When the temperature reaches the set stop temperature of 6°C, the refrigeration relay releases and stops refrigeration.
[0079] The process of humidity control is specifically as follows:
[0080] When the humidity in the film carrier box 13 is lower than the set humidity of 92%, the humidification relay closes. The positive pole of the ultrasonic humidifier 5 is connected to the positive pole of the power supply 1 through the relay, and the ultrasonic humidifier 5 is powered on to work. When it reaches the set humidity of 96%, the relay releases and stops humidification.
[0081] Preferably, a timer is added to the outer box 10, which starts timing when the antibody is dropped, i.e., when the antibody incubation begins.
[0082] Preferably, an Internet of Things two-dimensional code can also be preset on the outer box 10. By scanning the two-dimensional code, the user can obtain information such as the temperature, humidity, and incubation time of the incubation box.
[0083] Through the cooperation of the membrane loading box 13 and the hydrophobic isolation baffle 8, the present utility model can non-destructively separate the whole membrane carrying proteins at specific positions as required. On the premise of reducing the use of antibodies, irreversible damage to the membrane is avoided, and the purpose of simultaneously detecting multiple specific protein molecules can be achieved. On the basis of shortening the experimental period, both the economic benefit is improved and the working efficiency and accuracy of protein detection are enhanced.
[0084] At the same time, this incubation box also integrates a temperature and humidity sensor, a temperature control module, a humidification module, a timing module, and an Internet of Things two-dimensional code. It can timely adjust the temperature and humidity of the intelligent incubation box and perform accurate timing, so as to ensure the repeatability and accuracy between different batches of experiments. At the same time, the high-humidity environment inside the incubation box can reduce evaporation, thereby reducing the usage amount of specific antibodies and improving economic benefits. At the same time, it can be interconnected with the mobile phone by identifying the two-dimensional code. During the overnight incubation of antibodies for 12 - 16 hours, the temperature and humidity conditions of the incubation process can be remotely and dynamically monitored, and the experimental conditions can be recorded.
[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present utility model and are not intended to limit them. Although the present utility model has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that: modifications or equivalent replacements can still be made to the specific implementation manners of the present utility model. Any modification or equivalent replacement that does not depart from the spirit and scope of the present utility model shall be covered by the protection scope of the claims of the present utility model.
Claims
1. An incubation box for protein immunoblot antibody incubation, characterized in that, It includes an outer box (10) and a film carrier box (13). The film carrier box (13) is arranged in the outer box (10) to form a double-layer box structure; Multiple support plates (14) are provided in the outer box (10). The film carrier box (13) is placed on the support plates (14), and the support plates (14) are used to support the film carrier box (13); The bottom plate of the film carrier box (13) serves as a working substrate (15). A plurality of clamping members (9) are provided in the film carrier box (13), and each clamping member (9) is used to place a hydrophobic isolation baffle (8); During antibody incubation, a hydrophobic isolation baffle (8) is pressed on the detection film as needed on the working substrate (15) to divide the detection film into multiple detection areas; A humidity-preserving medium is filled in the area below the working substrate (15).
2. The incubation box for protein immunoblot antibody incubation according to claim 1, wherein, The shell of the film carrier box (13) is covered with a heat-insulating layer.
3. The incubation box for protein immunoblot antibody incubation according to claim 1, wherein A plurality of water through holes are provided on the support plates (14).
4. A incubation box for protein immunoblot antibody incubation according to claim 1, characterized in that, The width of the film carrier box (13) is smaller than that of the outer box (10).
5. A incubation box for protein immunoblot antibody incubation according to claim 1, characterized in that, Both the working substrate (15) and the hydrophobic isolation baffle (8) are made of hydrophobic materials.
6. The incubation box for protein immunoblot antibody incubation according to claim 1, wherein, The working substrate (15) is a corrugated board.
7. A incubation box for protein immunoblot antibody incubation according to claim 1, wherein, The hydrophobic isolation baffle (8) is placed directly above the support plates (14).
8. A incubation box for protein immunoblot antibody incubation according to claim 1, characterized in that, A digital constant-temperature temperature controller (4) and a digital humidity controller (2) are provided in the film carrier box (13); A display screen (3) is correspondingly arranged on the outer box (10). The display screen (3) is electrically connected to the digital humidity controller (2) and the digital constant-temperature temperature controller (4) to display the humidity and temperature in the film carrier box (13).
9. The incubation box for protein immunoblot antibody incubation according to claim 1, characterized in that, An ultrasonic humidifier (5), a heater (6) and a thermoelectric cooler (7) are provided in the outer box (10). The ultrasonic humidifier (5), the heater (6) and the thermoelectric cooler (7) are all equipped with fans.
10. The incubation box for protein immunoblot antibody incubation according to claim 1, wherein, A timing module is provided on the outer box (10); Or / and an Internet of Things two-dimensional code is provided on the outer box (10).