Non-protein confining liquid based on poloxamer and application of non-protein confining liquid in protein detection

Abstract

The invention relates to the technical field of protein detection, in particular to non-protein confining liquid based on poloxamer and application of the non-protein confining liquid in protein detection. The confining liquid for protein detection provided by the invention does not contain protein, and comprises 2-10g / L of a polyether compound; a nonionic surfactant with a volume fraction of 0.1%-0.2%; the volume fraction of the antibacterial preservative is 0.2%-0.5%; the balance is a buffer solution; the confining liquid has the advantages of being short in confining time, stable in confining effect, low in background signal, high in signal-to-noise ratio, simple to prepare, good in compatibility with detected antibodies and antigens, wide in applicable detection platform, simple and convenient to prepare, low in cost and the like, does not contain toxic components such as sodium azide and the like, and is suitable for large-scale production. And the method is suitable for the closing step of the solid-phase carrier in various protein detection experiments such as Western blot and ELISA (Enzyme-Linked Immunosorbent Assay).

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, and in particular to a non-protein blocking solution based on poloxamer and its application in protein detection, suitable for the blocking step in experiments such as Western blot, ELISA, and immunohistochemistry. Background Technology

[0002] In protein detection experiments, the blocking step is a crucial step in ensuring accurate detection results and reducing non-specific background interference. Its main function is to non-specifically block unbound regions on the solid-phase support during protein detection, preventing non-specific binding and thus improving the specificity and sensitivity of the detection.

[0003] Traditional blocking solutions often use reagents containing proteins, such as bovine serum albumin (BSA) and skim milk powder. These proteins have complex origins and suffer from problems such as large batch-to-batch variability, poor stability, difficulty in long-term storage, and excessively long blocking times. Furthermore, protein-based blocking solutions may generate high background signals on some detection platforms, reducing the signal-to-noise ratio and thus affecting detection sensitivity. Moreover, their compatibility is relatively poor, and they may not be suitable for various detection platforms and membrane materials. To overcome these problems, researchers have gradually explored protein-free blocking solutions.

[0004] Currently, most protein-free blocking solutions on the market use a variety of polymers to achieve a comprehensive blocking effect. Although some of them are superior to traditional blocking solutions, they have drawbacks in practical applications, such as high commercial prices, complex and variable formulations, and limited applicable scenarios.

[0005] Therefore, with the continuous development of protein detection technology and its increasingly wide range of applications, the demand for a fast, stable, protein-free, easy-to-prepare, and high-performance blocking solution is becoming increasingly urgent. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a rapid, stable, protein-free, and easy-to-prepare blocking solution formulation for non-specific blocking of unbound regions on a solid-phase support during protein detection, thereby overcoming the technical and application defects of existing technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a non-protein blocking solution based on poloxamer, wherein each liter of the non-protein blocking solution contains the following components at a final concentration:

[0009] Polyether compounds: 2-10 g / L;

[0010] Nonionic surfactants: volume fraction 0.1%-0.2%;

[0011] Antibacterial and preservative agent: volume fraction 0.2%-0.5%;

[0012] The rest are buffer solutions;

[0013] The polyether compound used is poloxamer.

[0014] Preferably, the nonionic surfactant is Tween 20; the volume fraction of the nonionic surfactant is 0.1%.

[0015] Preferably, the antibacterial preservative is Proclean 300; the volume fraction of the antibacterial preservative is 0.2%.

[0016] Preferably, the buffer solution is a Tris-buffered saline (TBS) solution, obtained by dissolving the required components of 1 unit TBS buffer system in 1 L of deionized water and adjusting the pH to a suitable level with HCl.

[0017] Preferably, the pH of the poloxamer-based non-protein blocking solution is 7.2-7.6.

[0018] Secondly, the present invention provides a method for preparing the above-mentioned non-protein blocking solution based on poloxamer, comprising the following steps:

[0019] Add the polyether compound, nonionic surfactant, and antibacterial preservative to the buffer solution and stir until all components are fully dissolved to obtain the non-protein blocking solution.

[0020] Thirdly, the present invention provides the application of the above-mentioned non-protein blocking solution based on poloxamer in protein detection.

[0021] Preferably, the non-protein blocking solution is used to block Western blot or ELISA plates.

[0022] Ideally, the sealing time should be 5-10 minutes.

[0023] Compared with the prior art, the sealing liquid of the present invention has the following advantages:

[0024] This invention provides a blocking solution that does not contain bovine serum albumin (BSA), skim milk powder, or other blocking components derived from animal proteins. This avoids contamination and batch-to-batch variations introduced by animal-derived proteins, and the solution is stable and easy to prepare. It is suitable for various detection platforms and membrane materials, and can be used in Western blot, ELISA, immunohistochemistry, and other experiments. It blocks unbound sites on the surface of solid supports, reducing non-specific binding by forming a hydration layer that creates steric hindrance. It exhibits high blocking efficiency, low background signal, and high signal-to-noise ratio, effectively improving detection sensitivity. It has broad application prospects in scientific research, clinical practice, pharmaceuticals, and testing. Attached Figure Description

[0025] Figure 1 The sealing result is shown in Example 1, which is the sealing liquid obtained in Example 1.

[0026] Figure 2 The sealing results are shown in Comparative Example 1.

[0027] Figure 3 The sealing results are shown in Comparative Example 2.

[0028] Figure 4 The sealing results are shown in Comparative Example 3.

[0029] Figure 5 The sealing results are shown in Comparative Example 4.

[0030] In the above attached diagram, A: 293T; B: J2M; C: HepG2. Detailed Implementation

[0031] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0032] In this embodiment of the invention, the polyether compound is poloxamer, specifically Pluronic® F-127, PubChem number: 24751; the nonionic surfactant is Tween 20; the antibacterial preservative is Proclean 300; and the buffer solution is a TBS buffer system (1X in 1L ddH2O), with the formulation shown in Table 1 below.

[0033] Table 1

[0034]

[0035] In the embodiments of the present invention, unless otherwise specified, all raw materials are available on the market, and those skilled in the art can select them according to actual needs.

[0036] The present invention will now be described in detail.

[0037] Example 1:

[0038] A typical preparation method of the sealing liquid described in this invention is as follows:

[0039] Prepare 1X TBS working solution (pH 7.4) according to the molar concentration in Table 1 for later use.

[0040] Weigh each component according to the following proportions:

[0041] Pluronic® F-127 5g, Tween 20 0.1% (v / v), Proclean 300 0.2% (v / v); mix and add to 1L of 1X TBS working solution, stir until all components are fully dissolved to obtain 1X ready-to-use rapid protein-free blocking solution.

[0042] Example 2:

[0043] The concentration of Pluronic® F-127 was changed to 2 g / L, and the other conditions were the same as in Example 1, to prepare a rapid protein-free blocking solution.

[0044] Example 3:

[0045] The concentration of Pluronic® F-127 was changed to 10 g / L, and the other conditions were the same as in Example 1, to prepare a rapid protein-free blocking solution.

[0046] Comparative Example 1

[0047] Pluronic® F-127 was replaced with polyethylene glycol 20000 (PEG20000) at a concentration of 5 g / L, and the other conditions were the same as in Example 1, to prepare a rapid protein-free blocking solution.

[0048] Comparative Example 2

[0049] Pluronic® F-127 was replaced with sodium dodecyl sarcosinate (SLS) at a concentration of 15 g / L, and the other conditions were the same as in Example 1, to prepare a rapid protein-free blocking solution.

[0050] Comparative Example 3

[0051] A blocking solution was prepared by mixing 5% skim milk powder with 1L of 1X TBS buffer.

[0052] Comparative Example 4

[0053] The blocking solutions obtained in Example 1 and Comparative Examples 1-5 were tested using a commercially available 5-minute ready-to-use blocking solution (Qichun Biotechnology, product number: YWB0501) as follows:

[0054] SETP1: Normally cultured cell lines were lysed using RIPA buffer and quantified using BCA, with a fixed sample loading volume of 10 μg per well. Using the Yaxin Bio-SDS-PAGE gel kit (10%) (catalog number: PG112), following the manufacturer's instructions, an appropriate amount of the lower gel was poured into a clean gel casting plate, and anhydrous ethanol was slowly added as a pressing layer. The plate was allowed to stand at room temperature for 30 minutes to allow the lower gel to solidify. After solidification, the pressing layer was discarded, and the anhydrous ethanol was removed with absorbent paper. Then, an appropriate amount of the upper gel was poured in, and a 15-well, 1 mm comb was inserted. The plate was allowed to stand at room temperature for 30 minutes, then the casting rack was removed, and the plate was completely immersed in deionized water at 4°C overnight (generally 12 hours) for use the following day.

[0055] SETP2: Carefully add the sample to the loading wells. Run electrophoresis at a constant voltage of 60-80V. When the bromophenol blue indicator reaches the interface between the upper and lower gel layers, adjust the voltage to 120-150V. After electrophoresis, remove the gel plate, leaving the lower gel intact for whole-membrane transfer (total transfer). The samples used are 293T (human embryonic kidney cell line), J2M (human acute T-lymphoblastic leukemia cell line), and HepG2 (human liver cancer cell line).

[0056] SETP3: Cut a 0.45µm PVDF membrane (Millipore, catalog number: IPVH00010) according to the length and width of the lower gel. First, immerse the PVDF membrane in methanol to activate it for 5 minutes. Then, assemble the transfer system in the following order: transfer clamp (white) - sponge - filter paper - PVDF membrane - lower gel - filter paper - sponge - transfer clamp (black). Note: This transfer system must be free of air bubbles and must remain in the transfer solution throughout the process; it must not be allowed to dry out.

[0057] After completion, the transfer clamps are loaded into the transfer tank according to the red-to-red and black-to-black pairings, and the transfer is performed for 45 minutes with a constant current of 400mA.

[0058] SETP4: After the transfer is completed, block each membrane separately using the blocking solutions obtained in Example 1 and Comparative Examples 1-5 for 10 minutes.

[0059] SETP5: After blocking, there is no need to wash with TSBT. Add the appropriately diluted primary antibody (rabbit anti-H3) directly to the incubation chamber and incubate overnight at 4°C on a horizontal or rocker incubator at 20 rpm. The next day, wash with TBST at room temperature for 5 minutes each time, on a horizontal or rocker incubator at 80 rpm.

[0060] After the primary antibody incubation is completed, add the secondary antibody (HRP-labeled goat anti-rabbit IgG, dilution ratio 1 / 10000) diluted at an appropriate ratio to the incubation box, incubate at room temperature for 50 minutes, on a horizontal or rocker shaker at 40 rpm, and then wash with TBST at room temperature for 5 minutes each time, on a horizontal or rocker shaker at 80 rpm.

[0061] SETP6: Using ECL chemiluminescent solution for color development, the source-culture imager automatically adjusts the exposure time for imaging to obtain the most suitable immunoblotting results.

[0062] The results of the above embodiments were evaluated for immunoblotting quality according to specificity and non-specificity, and the results are shown in Table 2 (i.e., the higher the specificity of the target band, the lighter the non-specific band is preferred).

[0063] Table 2

[0064]

[0065] The above-described embodiments are merely specific implementations of the present invention, used to illustrate the technical solutions of the present invention, and are not intended to limit it. The scope of protection of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any person skilled in the art can still modify or easily conceive of changes to the technical solutions described in the foregoing embodiments within the technical scope disclosed in the present invention, or make equivalent substitutions for some of the technical features; and these modifications, changes, 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, and should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A non-protein blocking solution based on poloxamer, characterized in that, Each liter of non-protein blocking solution contains the following final concentrations of components: Polyether compounds: 2-10 g / L; Nonionic surfactants: volume fraction 0.1%-0.2%; Antibacterial and preservative agent: volume fraction 0.2%-0.5%; The rest are buffer solutions; The polyether compound used is poloxamer.

2. The non-protein blocking solution based on poloxamer according to claim 1, characterized in that, The nonionic surfactant used is Tween 20; the volume fraction of the nonionic surfactant is 0.1%.

3. The non-protein blocking solution based on poloxamer according to claim 1, characterized in that, The antibacterial preservative used is Proclean 300; the volume fraction of the antibacterial preservative is 0.2%.

4. The non-protein blocking solution based on poloxamer according to claim 1, characterized in that, The buffer solution used is a Tris-buffered salt solution.

5. The non-protein blocking solution based on poloxamer according to claim 1, characterized in that, The pH of the poloxamer-based non-protein blocking solution is 7.2-7.

6.

6. A method for preparing a non-protein blocking solution based on poloxamer as described in any one of claims 1-5, characterized in that, Includes the following steps: Add the prescribed amounts of polyether compound, nonionic surfactant, and antibacterial preservative to the buffer solution and stir until all components are fully dissolved to obtain the non-protein blocking solution.

7. The application of the poloxamer-based non-protein blocking solution as described in any one of claims 1-5 in protein detection.

8. The application according to claim 7, characterized in that, The non-protein blocking solution is used to block Western blot or ELISA assays.

9. The application according to claim 7, characterized in that, The closure time is 5-10 minutes.

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