Method for dyeing biopolymer gel by using low-frequency oscillation electric field

Abstract

The invention relates to the technical field of biology, in particular to a method for biopolymer gel dyeing through a low-frequency oscillation electric field. The method comprises the steps of balancing, dyeing and decolorizing. Wherein the liquids filled into the electrolytic tank in each step are different and are respectively equilibrium liquid, staining liquid and decoloring liquid. The problems that when biopolymer gel is dyed under the action of an electric field, the dyeing time of high-concentration gel is long, the dyeing effect is poor, and small-molecular-weight strips are lost are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for using a low-frequency oscillating electric field to stain biopolymer gel. Background Art

[0002] Polyacrylamide gel electrophoresis (PAGE) is an important electrophoresis technique that uses polyacrylamide gel as a supporting medium to separate biomacromolecules such as proteins, peptides, and nucleic acids. Staining the gel after electrophoresis is a key step in visualizing the separation of samples. Currently, there are two methods for staining gels: manual and automated. The basic principle of automated instrument staining is to place the gel between two electrodes and use the dye to move in the electric field formed between the two electrodes to allow the dye to enter the gel for staining. Of course, most instruments also use an electric field to remove excess dye from the gel for decolorization.

[0003] The staining of this type of instrument is fully automated, with excellent staining effect, good consistency, short time, and low probability of causing physical damage to the gel. However, there are still the following shortcomings:

[0004] 1. The dye penetrates into the gel only on one side and has no effect on the other side, which results in poor staining effect on gels with high polyacrylamide concentrations and increased staining time, especially for gels with concentrations above 15% and increased cross-linking.

[0005] 2. Some small molecule proteins or peptides are prone to move from the gel to the external liquid in a continuously applied fixed electric field. Especially for proteins less than 10KD, the loss rate is high, which often results in reduced band visibility.

[0006] Therefore, how to perform staining and visualization operations on gels loaded with biopolymers is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] The present invention aims to overcome the shortcomings of the prior art by providing a method for staining biopolymer gels using a low-frequency oscillating electric field. This method addresses the problems of long staining times, poor staining results, and loss of small molecular weight bands in biopolymer gels stained under electric field conditions.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] A method for staining biopolymer gel using a low-frequency oscillating electric field comprises the following steps:

[0010] (1) Balance; the balanced working fluid is the balance fluid;

[0011] (2) dyeing; the dyeing working liquid is a dyeing liquid;

[0012] (3) Decolorization; the decolorization working liquid is a decolorization liquid; the balancing, dyeing, and decolorization are carried out in an electrolytic cell that can implement a low-frequency oscillating electric field.

[0013] The power supply for the electrolyzer can realize a low-frequency oscillating electric field, the main characteristics of which are as follows.

[0014] 1. The low-frequency oscillating electric field is powered by a DC power supply, and an H-bridge or relay is used to implement the electric field polarity conversion.

[0015] 2. The current waveform of the low-frequency oscillating electric field is a square wave.

[0016] 3. The frequency of the low-frequency oscillating electric field is 0.001-1Hz.

[0017] 4. The implementation voltage of the low-frequency oscillating electric field is 1-220V.

[0018] 5. The implementation current of the low-frequency oscillating electric field is 0.01-10A.

[0019] The main features of the electrolytic cell used for dyeing operations are as follows:

[0020] 1. The electrolytic cell is mainly composed of two electrode plates and a cell body. The volume of the electrolytic cell is 0.1-5L. Due to the use of an oscillating electric field, the two poles are not specifically distinguished as anode and cathode.

[0021] 2. The two electrodes are made of the same material, including but not limited to titanium alloy plated with platinum, rhodium or ruthenium.

[0022] 3. The size of the two electrode plates depends on the gel used, generally ranging from 5×5 cm to 40×40 cm, and their shape is not limited to rectangle.

[0023] 4. The electrolytic cell is equipped with automatic liquid inlet and discharge devices for replacing dyeing liquid, decolorizing liquid and balancing liquid.

[0024] 5. The two electrode plates can be placed horizontally or vertically in the electrolytic cell.

[0025] The entire instrument utilizes automated control, allowing for pre-programmed or on-site programming to control balancing, liquid supply, electric field application, liquid drainage, and alarms during the dyeing and bleaching processes. Besides controlling the power on / off and polarity switching, automated control also includes timing, liquid drainage, and alarms. This technology is conventional in automated control and will not be described in detail here.

[0026] The specific process of implementing the entire plan is as follows:

[0027] Place the gel containing biological high-protein to be stained in the electrolytic cell used for staining, close the cell cover, select the preset program or the on-site program, start the instrument to perform the staining operation, and after the staining is completed, open the cell cover and take out the stained gel. The working liquids in the staining process are divided into balancing liquid, staining liquid, and decolorizing liquid:

[0028] Equilibration solution: mainly, but not limited to, fixative solutions containing acetic acid, trichloroacetic acid, phosphoric acid, perchloric acid, and sulfosalicylic acid.

[0029] Staining solution: Dyes include, but are not limited to, amido black, Coomassie Brilliant Blue G250, Coomassie Brilliant Blue R250, ginger yellow, Ponceau S, and cyanine dyes. Main ingredients include, but are not limited to, phosphoric acid, acetic acid, trichloroacetic acid, perchloric acid, and sulfonic acid hydrochloric acid. Auxiliary ingredients include, but are not limited to, ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, and polyethylene glycol. Main organic solvents include, but are not limited to, ethanol, methanol, ethyl acetate, isopropanol, and acetone.

[0030] Decolorizing solution: Main ingredients may include but are not limited to: phosphoric acid, acetic acid, trichloroacetic acid, perchloric acid, sulfosalicylic acid; auxiliary ingredients may include but are not limited to: ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, polyethylene glycol, etc. Main organic solvent components may include but are not limited to: ethanol, methanol, ethyl acetate, isopropanol, acetone, etc.

[0031] Preferably, the balancing solution in step (1) comprises one or more of a 1-20% v / v aqueous solution of acetic acid, a 1-20% v / v aqueous solution of trichloroacetic acid, a 1-20% v / v aqueous solution of phosphoric acid, a 1-20% v / v aqueous solution of perchloric acid, and a 1-20% w / v fixative of sulfosalicylic acid.

[0032] Preferably, the dyeing solution of step (2) comprises 0.1%-0.5% w / v of dye, one or more of the main ingredients 10-50% v / v of phosphoric acid, 10-50% v / v of acetic acid, 10-50% v / v of trichloroacetic acid, 10-50% v / v of perchloric acid, and 10%-50% w / v of sulfosalicylic acid, 1-10% w / v of auxiliary ingredients, 10-60% v / v of organic solvent, and the balance is water; the dye comprises one or more of amido black, Coomassie Brilliant Blue G250, Coomassie Brilliant Blue R250, ginger yellow, Ponceau S, and cyanine dyes; the auxiliary ingredients comprise one or more of ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, and polyethylene glycol; and the organic solvent comprises one or more of ethanol, methanol, ethyl acetate, isopropanol, and acetone.

[0033] Preferably, the decolorizing solution in step (3) comprises one or more of the main components of phosphoric acid 10-50% v / v, acetic acid 10-50% v / v, trichloroacetic acid 10-50% v / v, perchloric acid 10-50% v / v, and sulfosalicylic acid 10%-50% w / v, an auxiliary component 1-10% w / v, an organic solvent 10-60% v / v, and the balance is water; the auxiliary component comprises one or more of ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, and polyethylene glycol; and the organic solvent comprises one or more of ethanol, methanol, ethyl acetate, isopropanol, and acetone.

[0034] Preferably, the biopolymer gel is polyacrylamide gel.

[0035] The preset program covers the entire operation process, which is divided into balancing, dyeing, and decolorizing steps. The liquids filled into the electrolytic cell in each step are different, namely balancing liquid, dyeing liquid, and decolorizing liquid. The corresponding liquid is first flushed in at the beginning of each step, and the liquid is drained and replaced after each step. Each individual balancing, dyeing, and decolorizing step is set with a predetermined duration. During this duration, the voltage (current) is applied to the electrolytic cell. It does not specifically require a constant voltage or constant current, and it can be adjusted according to the specific situation. The main thing is that the applied electric field changes direction according to the preset frequency to achieve a low-frequency oscillating electric field mode.

[0036] It should be pointed out that due to the low frequency, the remaining time beyond the set duration is generally not re-counted into the alternation time of the next step, and during the step switching process, the entire electrolytic cell is disconnected.

[0037] The beneficial effects of the present invention are: solving the problems of long dyeing time and poor effect of high-concentration gel and loss of small molecular weight bands in the dyeing of biological polymer gels under the action of an electric field. The present application aims to cover the dyeing of a larger part of polyacrylamide gels. There are many types of polyacrylamide gels, and this is a type that is difficult to dye using ordinary electrolytic cells. The present solution uses a low-frequency oscillating electric field as the dyeing power. In the dyeing action space, the low-frequency oscillation of the electric field can be used to allow the dye to enter from both sides of the gel at different times and penetrate the gel. Compared with applying an electric field in a single direction, the time for the dye to enter the gel is shortened and the proportion is increased. Another important aspect is that for small molecular weight proteins (1-10KD

[0038] ) and peptides or nucleic acids, or other types of biomacromolecules. When a continuously applied electric field is applied, most of the sample will move toward the positive electrode, which will reduce the visualization of the sample and may even lead to complete loss in extreme cases. Applying a low-frequency oscillating electric field during the staining, destaining, and equilibration process can avoid such problems. The position of the biomacromolecules in the gel is relatively stable in the oscillating electric field because their displacement is offset by changes in the direction of the electric field. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the specific implementation equipment of low-frequency oscillating electric field polymer gel staining. DETAILED DESCRIPTION

[0040] The present invention will be further described in detail below with reference to the examples, but is not limited thereto.

[0041] Example 1:

[0042] like Figure 1 As shown, the electrolytic cell contains electrode plates A and B. The cell is powered by a power supply, with the two poles of the power supply connected to the two electrode plates. A relay is located between the two poles of the power supply, which switches the two poles at low frequencies to form an oscillating electric field. Here, a constant voltage setting of 50V is used, with a current range of 0.5-3A, an oscillation frequency of 0.02Hz, and an electric field direction change every 50 seconds.

[0043] The electrolytic cell has a volume of about 200 ml, with the upper end being the liquid inlet and the lower end being the liquid outlet. Both electrode plates are placed perpendicular to the horizontal line in the cell. 16.5% Tricine gel to be stained and cytokine EGF (about 6KD) are placed between the two electrode plates.

[0044] The preset operation steps are: equilibration time 100 seconds, staining time 200 seconds, and decolorization time 200 seconds.

[0045] During the electrolytic cell operation, put into gel earlier after the position between two electrode plates, start-up program, this moment, electrolytic cell charged into the acetate aqueous solution of equilibrium solution 5% (v / v), after charging, apply the electric field by A to B direction, after 50 seconds, relay changed electric field direction, applies the electric field by B to A direction, after applying 50 seconds, finish equilibrium process, get rid of liquid, carry out staining step, staining solution, staining solution component is 0.1% Coomassie Brilliant Blue G250 (w / v), 30% methyl alcohol, 30% acetate (v / v), the aqueous solution of 10% (w / v) ammonium sulfate, after charging into the electrolytic cell, apply the electric field by A to B direction, after 50 seconds, be converted to the electric field by B to A direction, carry out 4 conversions according to frequency in 200 seconds. Carry out decolorization step at last, the decolorizing solution component is 10% (v / v) acetate, 10% (v / v) methyl alcohol, 7% (w / v) ammonium sulfate, aqueous solution, similar to above mode, no longer repeat. After the decolorization step is completed, the staining is completed. After draining the liquid in the pool, the gel is taken out for visual observation. The bands are clear, sharp, and not diffuse.

[0046] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for staining biopolymer gels using a low-frequency oscillating electric field, characterized in that: The following steps are involved: (1) Balance; the balanced working fluid is the balance fluid; (2) dyeing; the dyeing working liquid is a dyeing liquid; (3) Decolorization; the decolorization working liquid is a decolorization liquid; the balancing, dyeing, and decolorization are carried out in an electrolytic cell that can implement a low-frequency oscillating electric field.

2. The method according to claim 1, characterized in that The balancing solution in step (1) comprises one or more of a 1-20% v / v aqueous solution of acetic acid, a 1-20% v / v aqueous solution of trichloroacetic acid, a 1-20% v / v aqueous solution of phosphoric acid, a 1-20% v / v aqueous solution of perchloric acid, and a 1-20% w / v fixative of sulfosalicylic acid.

3. The method according to claim 1, characterized in that The dyeing solution of step (2) comprises 0.1%-0.5% w / v of dye, one or more of the main ingredients 10-50% v / v of phosphoric acid, 10-50% v / v of acetic acid, 10-50% v / v of trichloroacetic acid, 10-50% v / v of perchloric acid, and 10%-50% w / v of sulfosalicylic acid, 1-10% w / v of auxiliary ingredients, 10-60% v / v of organic solvent, and the balance is water; the dye comprises one or more of amido black, Coomassie Brilliant Blue G250, Coomassie Brilliant Blue R250, ginger yellow, Ponceau S, and cyanine dyes; the auxiliary ingredients comprise one or more of ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, and polyethylene glycol; and the organic solvent comprises one or more of ethanol, methanol, ethyl acetate, isopropanol, and acetone.

4. The method according to claim 1, wherein The decolorizing solution in step (3) comprises one or more of the following main components: 10-50% v / v phosphoric acid, 10-50% v / v acetic acid, 10-50% v / v trichloroacetic acid, 10-50% v / v perchloric acid, and 10%-50% w / v sulfosalicylic acid; 1-10% w / v auxiliary components; 10-60% v / v organic solvent; and the balance is water; the auxiliary components comprise one or more of ammonium sulfate, ammonium ferric sulfate, aluminum sulfate, urea, guanidine hydrochloride, and polyethylene glycol; and the organic solvent comprises one or more of ethanol, methanol, ethyl acetate, isopropanol, and acetone.

5. The method according to claim 1, characterized in that The low-frequency oscillating electric field is powered by a DC power supply, and an H-bridge or a relay is used to implement polarity conversion of the electric field.

6. The method according to claim 1, wherein The current waveform of the low-frequency oscillating electric field is a square wave, the frequency is 0.001-1 Hz, the implementation voltage is 1-220 V, and the implementation current is 0.01-10 A.

7. The method according to claim 1, characterized in that The electrolytic cell comprises two electrode plates and a cell body. The electrolytic cell is provided with automatic liquid inlet and outlet devices for replacing dyeing liquid or decolorizing liquid and balancing liquid.

8. The method according to claim 7, characterized in that The two electrode plates are made of the same material, which is titanium alloy plated with platinum, rhodium or ruthenium; the two electrode plates are placed horizontally or vertically in the electrolytic cell.

9. The method according to claim 8, characterized in that The two electrode plates are rectangular, square, trapezoidal or circular; a liquid inlet is provided at the upper end of the electrolytic cell and a liquid outlet is provided at the lower end; the two electrode plates are placed in the cell perpendicular to the horizontal line, and the gel to be dyed is placed between the two electrode plates.

10. The method according to claim 1, characterized in that The electrolytic cell adopts automatic control, and a reserved program or an on-site setting program is written to control the liquid supply, electric field implementation, liquid discharge and alarm action of the balance, dyeing and decolorization processes.

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