Application of a blocking agent in protein-free blocking solution

Abstract

The present invention belongs to the field of protein detection technology, and discloses an application of a blocking agent in a protein-free blocking solution. The blocking agent component is any one of fatty alcohol polyoxyethylene ether and fatty amine polyoxyethylene ether, and the concentration range is between 0.1wt% and 5wt%. The blocking solution is a ready-to-use blocking solution, which is easy to use and quick, does not require the addition of any additional reagents, works quickly and efficiently, has a short blocking time of only 5-10 minutes, does not contain protein components, has a low background after blocking, and has a high signal-to-noise ratio. The blocking solution is added with a preservative that does not affect the activity of HRP and AP, does not interfere with the detection of HRP or AP-labeled secondary antibodies, and does not interfere with biotin-based detection because it does not contain biotin.

Description

Technical Field

[0001] The invention belongs to the technical field of protein detection, and particularly relates to application of a blocking agent in a protein-free blocking solution. Background Art

[0002] Western blotting (WB), also known as immunoblotting, is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics. WB involves transferring proteins from a gel after electrophoresis separation to a solid support, such as a nitrocellulose membrane (NC membrane) or polyvinylidene fluoride (PVDF membrane), and then using specific antibodies to detect a specific antigen. It is now widely used in a variety of fields, including gene expression research at the protein level, antibody activity detection, and early disease diagnosis.

[0003] Enzyme-linked immunosorbent assay (ELISA) refers to a qualitative and quantitative detection method that combines soluble antigens or antibodies with solid phase carriers such as polystyrene and uses the specific binding of antigens and antibodies to conduct immune reactions.

[0004] In WB experiments, total protein is transferred from the gel to the solid support NC membrane or PVDF membrane through electricity. This is an adsorption effect of the membrane on the protein. The mechanism of this adsorption effect is a comprehensive effect including hydrophobic effect, electrostatics, van der Waals force, etc., which are all non-specific. During blocking, the blocking liquid is not only adsorbed to the blank position on the membrane, but also produces a competitive elution effect with the existing proteins on the membrane (target protein and miscellaneous proteins), thereby reducing the non-specific band signal. In ELISA experiments, when the concentration of the coated protein is too low, the surface of the solid support cannot be completely covered. Blocking is to allow a large amount of unrelated macromolecular substances to fill the gaps to prevent interfering substances from being adsorbed to the surface of the solid support in subsequent steps, resulting in non-specific color development. Therefore, it is necessary to use a blocking liquid to block unbound sites to avoid cross-reaction with antibodies or detection reagents.

[0005] Blocking is a crucial part of Western Blot, so choosing the right blocking solution allows antibodies to bind to antigens more specifically. Commonly used blocking solutions include skim milk powder, bovine serum albumin (BSA), casein, fish gelatin, and whole serum. Skim milk powder has a complex composition and is a mixture of many proteins, which can produce a lot of background signals. It also contains a small amount of biotin and alkaline phosphatase residues. When using secondary antibodies labeled with horseradish peroxidase (HRP) and alkaline phosphatase (AP), this can also lead to high background or increased background levels. BSA may contain contaminants such as antibodies or other serum proteins, which can cross-react with mammalian antibodies and increase nonspecific background signals. Summary of the Invention

[0006] In order to overcome the shortcomings of the existing technology, the present invention provides a blocking agent for use in a protein-free blocking solution. The protein-free blocking solution prepared using the blocking agent can complete blocking within 5-10 minutes, and is a non-protein high molecular substance that will not interfere with the detection of the target protein during detection.

[0007] The above object of the present invention is achieved by the following technical solution: a blocking agent is used in a protein-free blocking solution, wherein the blocking agent component is any one or two of fatty alcohol polyoxyethylene ether and fatty amine polyoxyethylene ether, and the concentration range is between 0.1wt% and 5wt%, wherein the molecular formula of the fatty alcohol polyoxyethylene ether is RO(CH2CH2O) n H, n≤100, its molecular structure is The molecular formula of fatty amine polyoxyethylene ether is RN[(CH2CH2O) n H]2, n≤100, its molecular structure is:

[0008] Another object of the present invention is to protect the protein-free blocking solution prepared from the blocking agent, which comprises a blocking agent, a pH buffer, a preservative, and water.

[0009] Furthermore, the protein-free blocking solution also includes one or both of inorganic salts sodium chloride and potassium chloride, with a concentration range of 0.01 mol / L-0.5 mol / L.

[0010] Furthermore, the protein-free blocking solution also includes a surfactant Tween-20, with a concentration ranging from 0.2% to 0.8%.

[0011] Furthermore, the pH buffer component is any one of phosphate and tris(hydroxymethyl)aminomethane, and the concentration range is between 0.01 mol / L and 0.5 mol / L.

[0012] Furthermore, the preservative component includes any one of Proclin 300 and sodium azide, with a concentration range of 0.01%-0.05%.

[0013] Another object of the present invention is to protect the use of the above-mentioned protein-free blocking solution, including its use in protein immunoblotting and enzyme-linked immunosorbent assays.

[0014] Compared with the prior art, the present invention has the following advantages: 1. nonionic surfactants (fatty alcohol polyoxyethylene ethers or fatty amine polyoxyethylene ethers, Tween-20) and inorganic ions (such as K+, Na+, H+, OH- and Cl-) are selected to reduce nonspecific signals in WB / ELISA experiments; 2. the blocking solution is a ready-to-use blocking solution that is convenient and quick to use and does not require the addition of any additional reagents; 3. the operation is fast and efficient, with a short blocking time of only 5-10 minutes; 4. the blocking solution does not contain protein components, resulting in low background after blocking and a high signal-to-noise ratio; 5. a preservative that does not affect the activity of HRP and AP is added to the blocking solution, and will not interfere with the detection of HRP- or AP-labeled secondary antibodies. At the same time, since it does not contain biotin, it will not interfere with biotin-based detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0016] Figure 1 This is a graph showing the western blot verification results of Example 1 without protein blocking solution 1;

[0017] Figure 2 This is a graph showing the western blot verification results in Example 1 without protein blocking solution 2;

[0018] Figure 3 This is a graph showing the western blot verification results in Example 2 without protein blocking solution 1;

[0019] Figure 4 This is a graph showing the western blot verification results in Example 2 without protein blocking solution 2. DETAILED DESCRIPTION

[0020] The present invention is described in detail below by specific examples, but the scope of protection of the present invention is not limited. Unless otherwise specified, the experimental methods adopted in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0021] Example 1

[0022] Verification of NC membrane transfer and blocking effect in Western Blot experiments

[0023] 1. Preparation of Blocking Solution

[0024] (1) Prepare protein-free blocking solutions according to Tables 1 and 2, respectively, and store at 4°C until ready for use.

[0025] Table 1 Ingredients of Protein-free Blocking Solution 1

[0026] Protein-free blocking buffer 1 component Component content Fatty alcohol polyoxyethylene ether 0.3% (W / V) Phosphate 0.015mol / L Sodium chloride 0.03mol / L potassium chloride 0.025mol / L; Tween-20 0.5% (W / V) Proclin300 0.05% (W / V)

[0027] Table 2 Composition of Protein-free Blocking Solution 2

[0028] Protein-free blocking buffer 2 components Component content Fatty amine polyoxyethylene ether 0.35% (W / V) Tris(hydroxymethyl)aminomethane 0.02mol / L Sodium chloride 0.04mol / L Tween-20 0.4% (W / V) Proclin300 0.02% (W / V)

[0029] (2) The reference blocking solution uses Beyotime's QuickBlock TM Western Blot experiments were performed according to the instructions using Western blocking buffer (Cat. No. P0252) and Western Blot skim milk powder blocking buffer (Dalian Meilun Biotechnology Co., Ltd., Cat. No. MA0097) for later use.

[0030] 2. Collect protein samples

[0031] Before collecting protein samples, dissolve the RIPA lysis buffer and mix thoroughly. For HeLa cells in a 6-well plate system, remove the culture medium and wash once with PBS. Add 200 μL of lysis buffer to each well (add PMSF within 30 minutes before use to a final PMSF concentration of 1 mM). Pipet several times to ensure adequate contact between the lysis buffer and cells. After 30 seconds, harvest the cells with a cell scraper and centrifuge at 10,000-14,000 g for 3-5 minutes. Remove the supernatant and set aside. Determine the protein concentration of the sample using a BCA protein assay kit.

[0032] 3. Electrophoresis

[0033] Use the PAGE gel ultra-fast preparation kit to prepare 10% SDS-PAGE gel. Add 5X SDS-PAGE protein loading buffer to the collected protein sample and heat it at 100°C or in a boiling water bath for 3-5 minutes; use a protein electrophoresis apparatus (Bio-rad) and add the corresponding volume of electrophoresis buffer according to the indicated scale before preparing for sample loading. After cooling to room temperature, load the sample directly into the SDS-PAGE gel loading well. Set the voltage to 70V, start electrophoresis for 1 hour, increase the voltage to 90-120V after the sample runs onto the separation gel, and continue electrophoresis. After the electrophoresis is completed, remove the stacking gel;

[0034] 4. Transfer

[0035] Soak the sponge, filter paper, and NC membrane in the transfer solution. Gently place the glue in the pre-cooled transfer solution. Place the black transfer splint on top and the white one on the bottom. First place a layer of sponge, then filter paper, then place the glue on the filter paper, and then the NC membrane on the glue. Then continue to place the filter paper and sponge symmetrically. Finally, clamp the splint, black against black, white against red, and insert it into the transfer tank (black glue and white membrane). Adjust the voltage to 300mA and the transfer time for 120 minutes.

[0036] 5. Closed

[0037] The protein-free blocking solution and the control blocking solution prepared above were blocked for 10 minutes, but the blocking time of the skimmed milk powder blocking solution in step 1 was 1 hour.

[0038] 6. Primary Antibody Incubation

[0039] The primary antibody (β-Actin antibody) was added at a dilution ratio of 1:1000 and reacted at room temperature for 2 hours.

[0040] 7. Secondary Antibody Incubation

[0041] The next day, the sections were washed three times with TBST at room temperature for 10 min each time. Goat anti-rabbit HRP-labeled secondary antibody (Abclonal, AS014) was added at a dilution ratio of 1:10,000, incubated at room temperature for 2 h, and washed three times with TBST for 10 min each time.

[0042] 8. Color rendering

[0043] use The color was developed using the Fectron ultrasensitive ECL luminescent solution and the digital chemiluminescence imaging system FusionFX6 multifunctional imaging system was used for exposure until the target band was clear. Figure 1 (Protein-free blocking solution 1), Figure 2 As shown in (Protein-free Blocking Solution 2), under identical sample and experimental conditions, the overall background after blocking with the protein-free blocking solution provided by the present invention was significantly lower than that after blocking with Biyuntian blocking solution, and the brightness of the target band was higher than that with skim milk powder. This demonstrates that the blocking solution provided by the present invention has a short blocking time and a high blocking effect without the influence of background color.

[0044] Example 2

[0045] The WB experiment was conducted to verify the sealing effect of PVDF membrane transfer. The solid phase carrier was changed to PVDF membrane (MILLIPORE) during transfer. The PVDF membrane was activated with methanol for 30s before transfer. Other operations were the same as in Example 1. Figure 3 (Protein-free blocking solution 1), Figure 4As shown in (protein-free blocking solution 2), the results are consistent with those in Example 1. The protein-free blocking solution provided by the present invention has a short blocking effect and a good blocking effect, which further illustrates that the present invention is compatible with NC membranes and PVDF membranes.

[0046] Example 3

[0047] Verification of the blocking effect in Elisa experiment

[0048] 1. Coating: Dilute PD-1 antigen to 1 μg / ml with coating solution, add 0.1 ml to each well, and incubate at 4°C overnight.

[0049] 2. Blocking: Prepare different blocking solutions according to the table below. Shake off the coating solution from the plate coated the day before and tap the plate. Add 3 different blocking solutions to 3 replicate wells, adding 0.2 ml to each well. Block at 37°C for 1 hour.

[0050] Blocking Buffer 1 Blocking Buffer 2 Blocking Buffer 3 Protein-free blocking buffer 1 5% BSA 5% skim milk powder

[0051] 3. Add test samples: discard the blocking solution and tap the plate; set the sample wells and add 100 μl of PD-1 antibody (SinoBiological, Catalog No. 10377-MM03) and test sample 1 (low concentration) and test sample 2 (high concentration) to the wells. Add only sample diluent to the blank wells and incubate at 37°C for 0.5 h.

[0052] 4. Add enzyme-labeled antibody: remove the antibody, wash the plate 3-5 times with PBST, tap the plate, and add enzyme-labeled secondary antibody (SinoBiological, product number SSA006) at a dilution of 1:10,000 (diluted with enzyme dilution buffer), 100 μl / well, and incubate at 37°C for 30 min.

[0053] 5. Color development: Remove the antibody, wash the plate 3-5 times with PBST, tap the plate, mix equal volumes of color development solution A and B, add 100 μl / well, and incubate at 37°C for 15 minutes in the dark. Add 50 μl of 2M sulfuric acid solution to each well to terminate the reaction; the reaction solution in the well will turn from blue to yellow.

[0054] 6. Reading: Measure the absorbance (OD) value of the solution in each well at 450 nm within 15 minutes after stopping the reaction.

[0055] The test results are shown in Table 3.

[0056] Table 3 Elisa verification results

[0057]

[0058] The results showed that the OD value of the blank wells of the plate coated with the protein-free blocking solution provided by the present invention was about 0.10, which was much lower than the OD value of the blank wells blocked with BSA and skimmed milk powder. Therefore, the protein-free blocking solution provided by the present invention can effectively reduce the background OD value and greatly shorten the blocking time.

[0059] The above-described embodiments are only preferred embodiments of the present invention, and are not intended to be all feasible embodiments of the present invention. Any obvious modifications made by a person skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A blocking agent for use as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that: The sealing agent component is fatty alcohol polyoxyethylene ether, wherein the molecular formula of fatty alcohol polyoxyethylene ether is RO(CH2CH2O) n H, n≤100, its molecular structure is The concentration of the sealing agent ranges from 0.1 wt% to 5 wt%.

2. Use of the blocking agent according to claim 1 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The protein-free blocking solution also includes one or both of inorganic salts sodium chloride and potassium chloride.

3. Use of the blocking agent according to claim 2 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The concentration of the inorganic salt is in the range of 0.01 mol / L to 0.5 mol / L.

4. Use of the blocking agent according to claim 1 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The protein-free blocking solution also includes a surfactant, Tween-20.

5. Use of the blocking agent according to claim 4 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The concentration of the surfactant Tween-20 is in the range of 0.2% to 0.8%.

6. Use of the blocking agent according to claim 1 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The pH buffer component is either phosphate or tris(hydroxymethyl)aminomethane, and the concentration range is between 0.01 mol / L and 0.5 mol / L.

7. Use of the blocking agent according to claim 1 as a protein-free blocking solution in protein immunoblotting or enzyme-linked immunosorbent assay, characterized in that The preservative ingredients include either Proclin 300 or sodium azide, with a concentration range of 0.01% to 0.05%.

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