Ice-bath-free protein transmembrane buffer solution and preparation method thereof

Abstract

The invention relates to the technical field of membrane transfer buffer solutions, and discloses an ice-bath-free protein membrane transfer buffer solution which comprises the following effective components: a complexing agent with the concentration of 1-3 mM, sodium hydrogen sulfite with the concentration of 0.5-1.5 mM, lauryl sodium sulfate with the mass percent of 0.1-0.2% and absolute ethyl alcohol with the volume percent of 10-30%, according to the membrane transfer buffer solution disclosed by the invention, a buffer group consisting of trihydroxymethyl aminomethane, beta-aminoethanesulfonic acid and 3-(N-morpholine) propanesulfonic acid is used, so that the buffer capacity of the electrophoresis buffer solution is remarkably improved, the membrane transfer time is shortened, and meanwhile, the relatively low heating efficiency can be maintained under the condition of relatively high current (voltage); ice bags, crushed ice and the like are not needed for cooling, so that the experiment efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of transfer buffer solutions, and more specifically, to a protein transfer buffer solution without ice bath and a preparation method thereof. Background Art

[0002] Protein immunoblotting (Western Blot, WB) is a method commonly used to detect the expression of specific proteins in the analyzed cells or tissues. It is a protein detection technique that transfers the total proteins of cells or tissues separated by electrophoresis from the gel to a solid support such as nitrocellulose (NC) membrane or polyvinylidene difluoride (PVDF) membrane, and then uses specific antibodies to detect a specific antigen. It is widely used in antibody activity detection, early disease diagnosis, and protein level expression research. The most commonly used method for protein transfer is electrophoresis transfer, which has the characteristics of being fast, effective, and maintaining high resolution of proteins in the gel. During the electrophoresis transfer process, the transfer buffer solution provides a medium for the electric field, allowing proteins to migrate from the negative electrode to the positive electrode under the action of the electric field force, and plays an important role in the protein transfer efficiency. Currently, the commonly used transfer buffer solutions are all based on the Tris-Glycine electrophoresis buffer system, including Towbin buffer solution (25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3) and Bjerrum Schafer-Nielsen buffer solution (48 mM Tris, 39 mM Glycine, 20% methanol, pH 9.2), etc.

[0003] Disadvantages of the prior art: As a traditional formula, it has wide applicability, but there are certain technical defects: First, the transfer speed is slow and the efficiency is low. It usually takes 1.5 - 2.5 hours, and the transfer efficiency of large molecular proteins is not high and cannot be completely transferred. Second, the formula contains methanol, a toxic reagent, which has a stimulating effect on the respiratory and gastrointestinal mucosa and a toxic effect on blood vessels and nerves. If not properly protected, it will cause harm to the experimenter. Third, the temperature of the electrophoresis solution significantly increases with the extension of the electrophoresis time. To ensure the transfer effect, an external ice bath is required to neutralize the heat generated during the transfer process. These technical defects result in a great increase in time cost and raw material cost, and pose a potential hazard to the experimenter's body, which does not meet the requirements of "safe experiment". Summary of the Invention

[0004] In order to solve the problems raised in the above background art, the present invention provides a protein transfer buffer solution without ice bath and a preparation method thereof.

[0005] The technical solution adopted by a protein transfer membrane buffer without ice bath provided by the present invention and its preparation method is as follows: A protein transfer membrane buffer without ice bath, the effective components of which include a complexing agent with a concentration of 1-3 mM, sodium bisulfite with a concentration of 0.5-1.5 mM, sodium dodecyl sulfate with a mass percentage of 0.1-0.2%, and absolute ethanol with a volume percentage of 10-30%.

[0006] Preferably, the buffer pair in the transfer membrane buffer includes tris(hydroxymethyl)aminomethane with a concentration of 45-75 mM, 2-aminoethanesulfonic acid with a concentration of 25-45 mM, and 3-(N-morpholino)propanesulfonic acid with a concentration of 40-60 mM.

[0007] Preferably, when the buffer pair is tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, the concentration ratio should be close to 1:1.

[0008] Preferably, the pH buffer of the transfer membrane buffer is composed of tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, 3-(N-morpholino), and propanesulfonic acid.

[0009] Preferably, the complexing agent includes disodium ethylenediaminetetraacetate.

[0010] Preferably, the transfer membrane condition of the transfer membrane buffer is a constant current of 400 mA to 500 mA.

[0011] Preferably, a preparation method of a protein transfer membrane buffer without ice bath includes the following steps:

[0012] (1) Weigh tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, disodium ethylenediaminetetraacetate, sodium bisulfite, and sodium dodecyl sulfate, and place them in a clean beaker;

[0013] (2) Add ultrapure water to the beaker and mix well using a magnetic stirrer;

[0014] (3) Add absolute ethanol with a volume percentage to the beaker and mix well with magnetic stirring;

[0015] (4) Make up the volume to 1 L with ultrapure water.

[0016] In summary, the present invention includes the following beneficial technical effects:

[0017] 1. By using a buffer group composed of tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, and 3-(N-morpholino)propanesulfonic acid, the transfer membrane buffer of the present invention significantly improves the buffering capacity of the electrophoresis buffer. While shortening the transfer membrane time, it can maintain a low heating efficiency under relatively large current (voltage) conditions, and does not require the use of ice bags, crushed ice, etc. to cool down, greatly improving the experimental efficiency.

[0018] 2. The transfer buffer of the present invention does not contain volatile methanol solution harmful to the human body, greatly improving safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic structural diagram of the comparison effect diagram of ice - bath - free protein transfer membrane and traditional protein transfer membrane in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] The following further elaborates on the present invention with reference to the Figure 1 accompanying drawings.

[0021] It should be noted that the drawings are schematic and not drawn to scale. For clarity and convenience in the figures, the relative sizes and proportions of the parts shown in the figures are exaggerated or reduced in size for illustration, and any dimensions are merely exemplary and not restrictive. Additionally, the same reference numerals are used for the same structures, elements, or fittings appearing in more than two figures to represent similar features.

[0022] An embodiment of the present invention discloses an ice - bath - free protein transfer buffer and its preparation method. Referring to Figure 1 , an ice - bath - free protein transfer buffer, the active ingredients of which include a complexing agent with a concentration of 1 - 3 mM. The complexing agent includes disodium ethylenediaminetetraacetate, sodium bisulfite with a concentration of 0.5 - 1.5 mM, sodium dodecyl sulfate with a mass percentage of 0.1 - 0.2%, and absolute ethanol with a volume percentage of 10 - 30%;

[0023] Through the above - mentioned structural design, ethanol is used instead of the commonly used toxic reagent methanol, making the use of the transfer buffer safer. During membrane transfer, the current and voltage will cause the temperature of the transfer buffer to rise, and the volatilization of ethanol can reduce the temperature of the transfer solution and reduce protein denaturation;

[0024] The addition of sodium bisulfite as a multifunctional auxiliary agent has the functions of anti - corrosion, antioxidant, and antibacterial

[0025] The addition of disodium ethylenediaminetetraacetate as a complexing agent can eliminate the interference of impurity metal ions such as calcium ions and potassium ions on membrane transfer;

[0026] The addition of sodium dodecyl sulfate can promote the elution of proteins from the gel, especially large - molecular - weight proteins that are difficult to elute, which is beneficial to improving the membrane transfer efficiency of macromolecular proteins;

[0027] The buffer pair in the transfer buffer includes tris (hydroxymethyl) aminomethane with a concentration of 45 - 75 mM, 2 - aminoethanesulfonic acid with a concentration of 25 - 45 mM, 3 - (N - morpholino) propanesulfonic acid with a concentration of 40 - 60 mM. When the buffer pair is tris (hydroxymethyl) aminomethane and 3 - (N - morpholino) propanesulfonic acid, the concentration ratio should be close to 1:1

[0028] The pH buffer group of the transfer membrane buffer solution consists of tris (hydroxymethyl) aminomethane, 2-aminoethanesulfonic acid, and 3-(N-morpholino) propanesulfonic acid;

[0029] Through the above structural design, the synergistic effect of the pH buffer group and ethanol enables the electrophoresis buffer solution to maintain a relatively lower heating efficiency during the transfer membrane process, thereby achieving the effect of eliminating the need for ice bath;

[0030] The addition of sodium dodecyl sulfate can promote the elution of proteins from the gel, especially large molecular weight proteins that are difficult to elute, which is beneficial to improving the transfer membrane efficiency of macromolecular proteins;

[0031] In the above transfer membrane buffer solution, the addition of disodium ethylenediaminetetraacetate as a complexing agent can eliminate the interference of impurity metal ions such as calcium ions and potassium ions on the transfer membrane;

[0032] In the above transfer membrane buffer solution, the addition of sodium bisulfite as a multifunctional auxiliary agent has the functions of anti-corrosion, anti-oxidation, and bacteriostasis;

[0033] Refer to Figure 1 (Comparison effect diagram of ice bath-free protein transfer membrane and traditional protein transfer membrane), the preparation method of the ice bath-free protein transfer membrane buffer solution, the transfer membrane condition is a constant current of 400 mA, and the steps are as follows:

[0034] (1), Weigh 45 mM tris (hydroxymethyl) aminomethane, 25 mM 2-aminoethanesulfonic acid, 40 mM 3-(N-morpholino) propanesulfonic acid, 1 mM disodium ethylenediaminetetraacetate, 0.5 mM sodium bisulfite, and 0.1% sodium dodecyl sulfate, and place them in a clean beaker;

[0035] (2), Add 700 mL of ultrapure water to the beaker and mix well using a magnetic stirrer;

[0036] (3), Add 10% by volume of absolute ethanol to the beaker and mix well with a magnetic stirrer;

[0037] (4), Make up the volume to 1 L with ultrapure water;

[0038] The preparation method of the ice bath-free protein transfer membrane buffer solution, the transfer membrane condition is a constant current of 450 mA, and the steps are as follows:

[0039] (1), Weigh 50 mM tris (hydroxymethyl) aminomethane, 35 mM 2-aminoethanesulfonic acid, 50 mM 3-(N-morpholino) propanesulfonic acid, 2 mM disodium ethylenediaminetetraacetate, 1 mM sodium bisulfite, and 0.15% sodium dodecyl sulfate, and place them in a clean beaker;

[0040] (2), Add 700 mL of ultrapure water to the beaker and mix well using a magnetic stirrer;

[0041] (3) Add anhydrous ethanol with a volume percentage of 20% to the beaker and mix well with magnetic stirring.

[0042] (4) Make up the volume to 1 L with ultrapure water.

[0043] A method for preparing a protein transfer membrane buffer without ice bath, the transfer membrane condition is a constant current of 500 mA, and the steps are as follows:

[0044] (1) Weigh 75 mM tris(hydroxymethyl)aminomethane, 45 mM β - aminoethanesulfonic acid, 60 mM 3-(N - morpholino)propanesulfonic acid, 3 mM disodium ethylenediaminetetraacetate, 1.5 mM sodium bisulfite and 0.2% sodium dodecyl sulfate, and place them in a clean beaker.

[0045] (2) Add 700 mL of ultrapure water to the beaker and mix well using a magnetic stirrer.

[0046] (3) Add anhydrous ethanol with a volume percentage of 30% to the beaker and mix well with magnetic stirring.

[0047] (4) Make up the volume to 1 L with ultrapure water.

[0048] Through the above structural design, the transfer membrane buffer uses a buffer group composed of tris(hydroxymethyl)aminomethane, β - aminoethanesulfonic acid and 3-(N - morpholino)propanesulfonic acid, which significantly improves the buffering capacity of the electrophoresis buffer. While shortening the transfer membrane time, it can maintain a low heat generation efficiency under relatively large current (voltage) conditions, and does not require the use of ice bags, crushed ice, etc. to cool down, thus greatly improving the experimental efficiency.

[0049] Finally, several points should be noted: First, in the description of the present invention, it should be noted that unless otherwise specified and limited, the terms "installation", "connection", "connection" should be understood in a broad sense, which can be mechanical connection or electrical connection, and can also be the communication inside two components. It can be directly connected. "Up", "down", "left", "right", etc. are only used to represent the relative position relationship. When the absolute position of the object being described changes, the relative position relationship may change;

[0050] Secondly: In the attached drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments of the present disclosure are involved. Other structures can refer to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other;

[0051] Finally: The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A protein transfer buffer without ice bath, characterized in that: Its active ingredients include a complexing agent with a concentration of 1 - 3 mM, sodium bisulfite with a concentration of 0.5 - 1.5 mM, sodium dodecyl sulfate with a mass percentage of 0.1 - 0.2%, and absolute ethanol with a volume percentage of 10 - 30%.

2. The ice-bath-free protein transfer buffer according to claim 1 and its preparation method are characterized in that: The buffer pair in the said membrane transfer buffer solution includes tris(hydroxymethyl)aminomethane with a concentration of 45 - 75 mM, 2-aminoethanesulfonic acid with a concentration of 25 - 45 mM, and 3-(N-morpholino)propanesulfonic acid with a concentration of 40 - 60 mM.

3. The protein transfer buffer without ice bath according to claim 2 and its preparation method are characterized in that: When the buffer pair is tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, the concentration ratio should be close to 1:

1.

4. A protein transfer buffer without ice bath and its preparation method according to claim 3, characterized in that: The pH buffering of the said membrane transfer buffer solution is composed of tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, 3-(N-morpholino), and propanesulfonic acid.

5. A protein transfer buffer without ice bath and its preparation method according to claim 4, characterized in that: The said complexing agent includes disodium ethylenediaminetetraacetate.

6. The ice-bath-free protein transfer buffer according to claim 5 and its preparation method are characterized in that: The membrane transfer condition of the said membrane transfer buffer solution is a constant current of 400 mA - 500 mA.

7. A protein transfer buffer without ice bath and its preparation method according to claims 1-6, characterized in that: A preparation method of an ice-bath-free protein membrane transfer buffer solution includes the following steps: (1) Weigh tris(hydroxymethyl)aminomethane, 2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, disodium ethylenediaminetetraacetate, sodium bisulfite, and sodium dodecyl sulfate, and place them in a clean beaker; (2) Add ultrapure water to the beaker and mix well using a magnetic stirrer; (3) Add absolute ethanol with a volume percentage to the beaker and mix well with magnetic stirring; (4) Make up the volume to 1 L with ultrapure water.

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