Rapid membrane transfer liquid suitable for western blot experiment

Abstract

The invention belongs to the technical field of immunobiochemistry, and relates to a rapid membrane transfer solution suitable for western blot experiments, which is prepared by the following steps: adding tris (hydroxymethyl) aminomethane, glycine, ethylenediamine tetraacetic acid and tris (hydroxymethyl) methylglycine into deionized water, stirring, mixing and dissolving at normal temperature, fixing the volume, adding ethanol, and uniformly mixing to obtain the membrane transfer solution. The trihydroxymethyl aminomethane, the glycine, the ethylenediamine tetraacetic acid and the tris (hydroxymethyl) methylglycine are compatible, the prepared film transfer liquid is reliable in film transfer result, clear and stable in developing result and not prone to forming crystals after being used for a long time, film transfer can be completed in a 25-v constant-pressure mode within a short time, the electricity transfer time is remarkably shortened, and the film transfer efficiency is improved. The immunoprotein imprinting efficiency is remarkably improved, little heat is generated in the membrane transferring process, the membrane transferring liquid does not contain volatile solution methanol harmful to the human body, the pH value does not need to be adjusted, and the method has the advantages of being easy and convenient to purchase raw materials, easy to prepare, environmentally friendly and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of immunobiochemistry, and relates to a transfer fluid in an immunoblotting transfer process, in particular to a rapid transfer fluid suitable for western blot experiments. Background Art

[0002] Western blotting (WB) is an important method in protein analytical chemistry. The proteins in the mixed antigen sample are separated according to their molecular weight by polyacrylamide gel electrophoresis, and then the antigen components are transferred to a solid phase carrier (blotting membrane) for solidification by membrane transfer technology (this process is called membrane transfer). Finally, the antigen-fixed blotting membrane is detected and analyzed using an immunoisotope probe or an immunoenzyme probe. However, this method has high requirements. In any step, even a small error may significantly change the subsequent links and cause deviations in the experiment. Therefore, researchers have invested a lot of time and energy to master this method, but they may not get satisfactory results.

[0003] At present, the transfer technology in the WB process is divided into wet transfer and dry transfer. Wet transfer has a good transfer effect on proteins of various molecular weights, and the transfer efficiency is high, but the operation time is long. Proteins with a molecular weight below 30kD need to be transferred for 30 minutes, 30-70kD need to be transferred for 60 minutes, and 70-140kD need to be transferred for 90-110 minutes. In addition, wet transfer consumes a lot of reagents and the operation is relatively complicated. A lot of heat will be generated during the experiment, and an ice bath environment needs to be provided. Dry transfer has a fast transfer speed, saves reagents, and the operation steps are relatively simple. It does not require a long time to wait for the transfer liquid to immerse, and the result background is clean, and the temperature control requirements are low. However, the scope of application of dry transfer is relatively narrow: it is generally more suitable for proteins with a molecular weight of less than 100kD, and the transfer effect for large molecular proteins may not be as ideal as wet transfer. Moreover, the cost of dry transfer instruments is relatively high, and usually a special dry transfer instrument is required. The instrument price is relatively high, and some dry transfer instruments are closed systems, which may limit the flexibility of the experiment to a certain extent. Due to price issues, wet transfer is chosen by more experimenters, but the disadvantages of wet transfer make the entire WB experiment too time-consuming, and the increase in operation steps also makes the results more prone to errors. Summary of the invention

[0004] The purpose of the present invention is to provide a rapid transfer solution suitable for western blot experiments. The transfer solution is compatible with existing commonly used transfer instruments, shortens the time required for transfer, is simple to prepare, safe and non-toxic, thus improving the success rate and productivity of the experiment and reducing the cost of the experiment. The transfer solution can completely replace the current common transfer solution.

[0005] The technical solution adopted by the present invention is:

[0006] The invention discloses a rapid transfer solution suitable for western blot experiments. The transfer solution is prepared by mixing and dissolving tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)methylglycine (Tricine) and deionized water, and then adding ethanol and mixing well. The final concentration of each component is: Tris 190-280mM, Gly 110-180mM, EDTA 1.4-2.0mM, Tricine 60-110mM, and the volume concentration of ethanol is 15-25%.

[0007] A method for preparing the above-mentioned rapid transfer solution suitable for western blot experiments, the steps of which are as follows:

[0008] Tris, glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) were added into deionized water according to the ratio, mixed and dissolved by stirring at room temperature, fixed to volume, and then ethanol was added and mixed to obtain transfer solution.

[0009] The invention prepares a transfer solution by combining tris(hydroxymethyl)aminomethane, glycine, ethylenediaminetetraacetic acid and tris(hydroxymethyl)methylglycine. The transfer solution has reliable transfer results, clear and stable development results, is not easy to form crystals after long-term use, and can complete the transfer in a short time, which significantly reduces the electrotransfer time and significantly improves the efficiency of immunoprotein imprinting. The transfer solution generates less heat during the transfer process, does not contain volatile solution methanol that is harmful to the human body, does not need to adjust the pH value, and has the characteristics of easy purchase of raw materials, simple preparation, environmental friendliness, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The transfer effect of the transfer solution of the embodiment of the present invention using eBLOT L1 for transfer. A: The effect of the transfer solution of Example 1 on the PVDF membrane after transfer by Ponceau red staining; B: The effect of the gel after transfer of Example 1 by Coomassie brilliant blue staining; C: The effect of the transfer solution of Example 2 on the PVDF membrane after transfer by Ponceau red staining; D: The effect of the gel after transfer of Example 2 by Coomassie brilliant blue staining; E: The effect of the transfer solution of Example 3 on the PVDF membrane after transfer by Ponceau red staining; F: The effect of the gel after transfer of Example 3 by Coomassie brilliant blue staining.

[0011] Figure 2 The transfer effect of the transfer solution of Example 1 of the present invention using Bio-Rad transfer. A: The PVDF membrane after transfer of the transfer solution of Example 1 was stained with Ponceau red; B: The gel after transfer of Example 1 was stained with Coomassie brilliant blue.

[0012] Figure 3 The transfer results of high-concentration transfer buffer using Bio-Rad transfer. A: PVDF membrane after transfer was stained with Ponceau red; B: Gel after transfer was stained with Coomassie brilliant blue.

[0013] Figure 4 The transfer results of eBLOT L1 transfer using low-concentration transfer buffer. A: PVDF membrane after transfer was stained with Ponceau red; B: Gel after transfer was stained with Coomassie brilliant blue.

[0014] Figure 5 The transfer results of low-concentration transfer buffer using Bio-Rad transfer. A: PVDF membrane after transfer was stained with Ponceau red; B: Gel after transfer was stained with Coomassie brilliant blue.

[0015] Figure 6 The results of the transfer using eBLOT L1 as the transfer solution in comparative example 3 are shown in Figure 3. A: The PVDF membrane after transfer was stained with Ponceau red; B: The gel after transfer was stained with Coomassie brilliant blue.

[0016] Figure 7 The results of the transfer solution of Comparative Example 3 using Bio-Rad transfer solution are shown in Figure 3. A: The PVDF membrane after transfer was stained with Ponceau red; B: The gel after transfer was stained with Coomassie brilliant blue.

[0017] Figure 8 The results of the transfer solution of Comparative Example 4 using Bio-Rad transfer solution are shown in Figure 4. A: The PVDF membrane after transfer was stained with Ponceau red; B: The gel after transfer was stained with Coomassie brilliant blue. DETAILED DESCRIPTION

[0018] The following examples are used to further describe the specific embodiments of the present invention. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available, with analytically pure reagents being preferred reagents.

[0019] 1. Preparation of transfer solution designed in this application

[0020] Embodiment 1

[0021] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and mix to dissolve at room temperature, fix the volume, then add ethanol and mix to obtain transfer solution. The final concentration of each component is: Tris 200mM, Gly 175mM, EDTA 1.4mM, Tricine 65mM, and the volume concentration of ethanol is 16%.

[0022] Embodiment 2

[0023] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and dissolve at room temperature, fix the volume, and then add ethanol and mix well to obtain transfer solution. The final concentration of each component is: Tris 240mM, Gly 120mM, EDTA 1.8mM, Tricine 105mM, and the volume concentration of ethanol is 22%.

[0024] Embodiment 3

[0025] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and dissolve at room temperature, fix the volume, and then add ethanol and mix well to obtain transfer solution. The final concentration of each component is: Tris 270mM, Gly 150mM, EDTA 1.7mM, Tricine 80mM, and the volume concentration of ethanol is 18%.

[0026] 2. Preparation of transfer buffer

[0027] Comparative Example 1: Preparation of high concentration transfer solution

[0028] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and dissolve at room temperature, fix the volume, and then add ethanol and mix well to obtain transfer solution. The final concentration of each component is: Tris 280mM, Gly 220mM, EDTA 2.2mM, Tricine 120mM, and the volume concentration of ethanol is 30%.

[0029] Comparative Example 2: Preparation of low concentration transfer solution

[0030] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), ethylenediaminetetraacetic acid (EDTA), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and mix to dissolve at room temperature, fix the volume, then add ethanol and mix to obtain transfer solution. The final concentration of each component is: Tris 150mM, Gly 110mM, EDTA 1.1mM, Tricine 60mM, and the volume concentration of ethanol is 10%.

[0031] Comparative Example 3: Preparation of transfer fluids with different components

[0032] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), and ethylenediaminetetraacetic acid (EDTA) into deionized water, stir and dissolve at room temperature, fix the volume, and then add ethanol and mix well to obtain transfer solution. The final concentration of each component is: Tris 200mM, Gly 160mM, EDTA 1.5mM, and the volume concentration of ethanol is 20%.

[0033] Comparative Example 4: Preparation of transfer fluids with different components

[0034] Add tris(hydroxymethyl)aminomethane (Tris), glycine (Gly), and tris(hydroxymethyl)methylglycine (Tricine) into deionized water, stir and mix to dissolve at room temperature, fix the volume, then add ethanol and mix well to obtain transfer solution. The final concentration of each component is: Tris 230mM, Gly 180mM, Tricine 90mM, and the volume concentration of ethanol is 20%.

[0035] 3. Verification of transfer effect

[0036] (I) Test instruments and methods:

[0037] 1. eBLOT L1 transfer apparatus. Transfer conditions: constant pressure 25V. Transfer protein: protein molecular weight 15kDa-220kDa, sample amount 20μg.

[0038] Transfer process: Use the transfer clip that comes with the eBLOT L1 instrument. Place the dry sponge, methanol-activated PVDF membrane, gel after electrophoresis, and dry sponge in the clip in the order of the positive pole (red button side) of the clip, symmetrically up and down, remove the bubbles and close the clip. Then put it into the instrument and select normal, long or short mode for transfer according to the program. The instrument automatically flushes the pure water pipeline, adds transfer solution and performs the transfer process.

[0039] Transfer result detection: 1. After staining the PVDF membrane with Ponceau red for 5 minutes and then rinsing it with pure water for 3 times, observe the number and molecular weight range of the bands transferred to the PVDF membrane. If the protein is successfully transferred to the PVDF membrane, red bands will be left, and the molecular weight of the protein can be inferred based on the rainbow protein marker. 2. After the gel is soaked in Coomassie Brilliant Blue dye for 10 minutes and rinsed with pure water for 3 times, observe the position and content of the protein remaining on the gel. If the protein remains on the gel, blue protein bands will be left, and the more residues, the darker the blue.

[0040] 2. Bio-Rad transfer apparatus. Transfer conditions: 400 mA, 20 min. Transfer protein: protein molecular weight 15kDa-220kDa, sample amount 20 μg.

[0041] Transfer process: Use the transfer clip and transfer tank provided with the instrument, soak the black side of the clip in the transfer solution, then put the sponge pad, three layers of filter paper, gel after electrophoresis, methanol-activated PVDF membrane, another three layers of filter paper, and sponge pad on the black side in turn, align them up and down, drive away the bubbles, and clamp the clip. Then align the black side of the clip with the black side of the transfer tank and put it into the tank, pour the transfer solution, cover the power cover, set the power supply to 400mA and transfer for 20 minutes.

[0042] Transfer result detection: Same as eBLOT L1 instrument.

[0043] (II) Results and analysis

[0044] ① The transfer solution obtained in Examples 1, 2, and 3 was transferred on the eBLOT L1 transfer instrument, and the normal, long, and short modes were selected respectively. The PVDF membrane after transfer was stained with Ponceau red and photographed. The results are as follows: Figure 1 As shown in A, C, and E, the gel after transfer was stained with Coomassie Brilliant Blue and photographed. Figure 1 As shown in B, D, and F.

[0045] Depend on Figure 1 It can be seen that the transfer solution provided in the present application can achieve good transfer effects in different modes. The protein bands are completely displayed on the PVDF membrane after being dyed red by Ponceau, and the bands with molecular weights ranging from 15 kDa to 220 kDa are displayed (such as Figure 1 The results are shown in A, C, and E of Figure 1, indicating that these proteins can be completely and clearly transferred to the PVDF membrane. There are basically no blue protein bands left on the gel after transfer, and only some slight lane colors remain (such as Figure 1 B, D, and F), indicating that the protein transfer was complete.

[0046] ② The transfer solution obtained in Example 1 was transferred on a Bio-Rad transfer instrument. The PVDF membrane after transfer was stained with Ponceau red and photographed. The results are as follows: Figure 2 As shown in A, the gel after transfer was stained with Coomassie Brilliant Blue and photographed. Figure 2 As shown in B.

[0047] Depend on Figure 2 It can be seen that a large number of Ponceau red-stained protein bands are displayed on the PVDF membrane after transfer, with molecular weights ranging from 15 kDa to 220 kDa, indicating that these proteins can be completely and clearly transferred to the PVDF membrane (such as Figure 2 After transfer, there is basically no blue protein band left on the gel, only some slight lane color remains, indicating that the protein transfer is complete (as shown in A). Figure 2 Moreover, the transfer time is 20 min, which is much shorter than the time required for traditional transfer experiments.

[0048] ③ Use high-concentration transfer solution (Comparative Example 1, the composition is consistent with that of the embodiment) and use two transfer instruments to transfer the membrane.

[0049] The membrane was transferred using the eBLOT transfer instrument (normal mode): the instrument reported an error after running for 10 minutes, the membrane transfer was terminated, the solution was overheated, the PVDF membrane was burnt yellow, there was a burnt smell, and the protein transfer was unsuccessful.

[0050] The membrane was transferred using a Bio-Rad transfer instrument (400 mA, 20 min). The transfer process generates a lot of heat. Figure 3 As shown, the results showed that the PVDF membrane was not transferred to the protein, and no red protein bands appeared (such as Figure 3 However, a large amount of protein remains on the gel after transfer, and the blue bands are deep and numerous (as shown in A). Figure 3 (as shown in B).

[0051] ④ Use a low-concentration transfer solution (Comparative Example 2, the composition is consistent with that of the embodiment) and use two transfer instruments to transfer the membrane.

[0052] The membrane was transferred using the eBLOT transfer instrument (normal mode). The results are as follows: Figure 4 As shown in the PVDF membrane staining results, the internal reference protein with a molecular weight of about 43 kDa was obviously lost (such as Figure 4 The results are shown in Figure A, indicating that low-concentration transfer buffer has poor transfer effect on low-molecular-weight proteins. In addition, the blue bands on the gel after transfer are numerous and obvious (as shown in Figure Figure 4 This indicates that a lot of high molecular weight proteins still remain on the gel and the membrane transfer is not successful.

[0053] The membrane was transferred using a Bio-Rad membrane transfer instrument (400 mA, 20 min). Figure 5 As shown, the results showed that no red protein bands appeared on the PVDF membrane, indicating that the protein did not bind to the PVDF membrane (such as Figure 5 However, there are still many blue bands on the gel after transfer, indicating that a large amount of protein remains (such as Figure 5 (as shown in B). The transfer effect is poor.

[0054] ⑤ Use transfer solutions with different compositions (Comparative Example 3) and use two transfer instruments to transfer the membranes.

[0055] The membrane was transferred using the eBLOT transfer instrument (normal mode). The results are as follows: Figure 6 As shown in the results, the PVDF membrane did not show obvious red protein bands in the molecular weight range above 100 kDa, indicating that the transfer effect of large molecular weight proteins was poor (e.g. Figure 6 However, there are still many blue bands on the gel after transfer, indicating that a large amount of protein remains (such as Figure 6 The overall transfer effect is poor.

[0056] The membrane was transferred using a Bio-Rad membrane transfer instrument (400 mA, 20 min). Figure 7 As shown in the results, no red protein bands appeared on the PVDF membrane, indicating that the protein was not bound (transferred) to the PVDF membrane (such as Figure 7 The transfer failed. However, after transfer, there were still many dark blue bands on the gel, indicating that a large amount of protein remained on the gel (as shown in A). Figure 7 (as shown in B). The transfer effect is poor.

[0057] ⑥ Using transfer solutions with different compositions (Comparative Example 4), two transfer instruments were used for transfer.

[0058] The membrane transfer was performed using the eBLOT membrane transfer instrument (normal mode). After the instrument was running, the solution generated excessive heat, which burned the sponge, gel and PVDF membrane, resulting in membrane transfer failure.

[0059] The membrane was transferred using a Bio-Rad membrane transfer instrument (400 mA, 20 min). Figure 8 As shown in the results, no red protein bands appeared on the PVDF membrane, indicating that the protein was not bound (transferred) to the PVDF membrane (such as Figure 8 After transfer, there are still many blue bands on the gel, indicating that a large amount of protein remains on the gel (as shown in A). Figure 8 (as shown in B). The transfer effect is poor.

[0060] The results show that the transfer solution prepared by the present invention by combining tris(hydroxymethyl)aminomethane, glycine, ethylenediaminetetraacetic acid and tris(hydroxymethyl)methylglycine does not contain volatile solution methanol that is harmful to the human body, is not easy to form crystals after long-term use, is suitable for a variety of transfer instruments, does not need to adjust the pH value during the transfer process, generates less heat, and can complete the transfer in a short time, which can significantly reduce the experimental time, shorten the time of the traditional transfer experiment to 11-21 minutes, and significantly improve the efficiency of immunoprotein imprinting. The results of Ponceau red staining and Coomassie brilliant blue staining after transfer confirm that the transfer solution has high transfer efficiency, less protein residue, reliable results, clear and stable development results, and has the characteristics of easy purchase of raw materials, simple preparation, environmental friendliness, etc.

[0061] Given that NC membrane and PVDF membrane have similar action principles in protein binding in transfer experiments, the transfer solution of this application is also applicable to NC membrane.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rapid transfer solution suitable for western blot experiments, characterized in that: The method comprises mixing and dissolving tris(hydroxymethyl)aminomethane, glycine, ethylenediaminetetraacetic acid, tris(hydroxymethyl)methylglycine and deionized water, and then adding ethanol and mixing well to obtain a transfer solution, wherein the final concentration of each component is: Tris 190-280mM, Gly 110-180mM, EDTA 1.4-2.0mM, Tricine 60-110mM, and the volume concentration of ethanol is 15-25%.

2. A method for preparing a rapid transfer solution suitable for western blot experiments according to claim 1, characterized in that: The steps are as follows: Tris(hydroxymethyl)aminomethane, glycine, ethylenediaminetetraacetic acid and tris(hydroxymethyl)methylglycine were added into deionized water according to the proportion, mixed and dissolved under stirring at room temperature, fixed to volume, and then ethanol was added and mixed to obtain transfer solution.

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