CN114544738A - a running buffer - Google Patents

Abstract

The invention provides an electrophoresis buffer, which is prepared by adjusting the pH to 8.3-8.6 from the following raw materials: tris(hydroxymethyl)aminomethane, disodium EDTA, NaCl and distilled water. The electrophoresis buffer prepared by the invention is subjected to the electrophoresis experiment of serum cellulose acetate film, and the probability of occurrence of five lines is high, the zones are clearly visible, and the zones are obvious; the ionic strength of the buffer prepared by the invention is low, it is an alkaline buffer, and the pH of the buffer is easy Adjustable, easy to calculate ionic strength, suitable for all electrophoresis experiments in place of barbiturate-barbital sodium. In addition, the electrophoresis buffer prepared by the present invention can also be used in other life science experiments with different pH and different ionic strengths as required.

Description

a running buffer

technical field

The invention belongs to the technical field of biochemistry, in particular to an electrophoresis buffer.

Background technique

Biochemistry is an important professional basic course for medicine, animal medicine and production majors in general colleges and universities, and it is an experiment-based subject. The experimental teaching focuses on the qualitative and quantitative analysis techniques of macromolecular substances. One of the important and basic experimental techniques is electrophoresis.

In the electrophoresis experiment, cellulose acetate film electrophoresis is simple, intuitive and practical, with the characteristics of small electroosmosis, fast separation speed, clear zone, simple operation and small sample consumption. One of the most basic teaching programs in This method utilizes proteins as ampholytes. In a certain pH environment, the amount of charge on the surface is different. In addition, the size and shape of the particles are also different. That is, under the condition of electrification, the migration speed in the electric field is different. The higher the voltage in the electric field, the electrode is charged. The greater the force of the particle, the faster the particle's swimming speed, but the voltage is also affected by the length of the supporting medium, and the moving speed of the charged particle is proportional to the voltage difference per unit length on the supporting medium, that is, proportional to the potential gradient, so that Different proteins can be separated. In recent years, two drugs, barbiturate and barbital sodium, are not easy to buy. On the one hand, the factors are due to the strict control of special drugs, and on the other hand, the demand for experimental drugs is large and the purchase amount is limited. Furthermore, the barbiturate-sodium barbital buffer is easy to form crystals at both ends of the electrode during the experiment, which affects the electrophoresis efficiency, making it difficult to judge the positive result.

Serum protein electrophoresis has broad application prospects. Serum protein acetate membrane electrophoresis can separate serum proteins into five distinct zones: albumin, α1-globulin, α2-globulin, and β-globulin. protein and gamma-globulin. Abnormal changes in a certain serum protein can be used to infer whether certain diseases have occurred. For example, the level of serum globulin has a certain influence on the levels of inflammatory factors and neurohormones in patients with acute ischemic stroke after neurointervention. Serum globulin The abnormal increase may also have a certain relationship with the occurrence of multiple myeloma. Multiple myeloma secretes abnormal monoclonal immunoglobulin (M protein) resulting in increased abnormal globulin and decreased normal globulin synthesis. Therefore, the need to develop a running buffer is particularly important.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a kind of electrophoresis buffer solution in view of the above-mentioned deficiencies of the prior art. The electrophoresis buffer solution prepared by the invention has a high probability of occurrence of five zone results, and NaCl is added to effectively reduce the electroosmotic phenomenon. At the same time, the addition of NaCl makes the ionic strength of the buffer system increase more suitable for electrophoresis experiments.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: a kind of electrophoresis buffer, which is prepared by adjusting pH to 8.3-8.6 from the following raw materials: tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and Distilled water.

Preferably, the dosage ratio of the tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water is 10.800g:0.930g:1.317g:1L.

Preferably, a nitric acid solution is used to adjust the pH.

Preferably, the mass fraction of the nitric acid solution is 50%.

The present invention also provides a method for preparing the above electrophoresis buffer. Tris-alkali (tris-base) with a mass of 10.800 g, disodium EDTA with a mass of 0.930 g and NaCl with a mass of 1.317 g are added in a volume of Fully dissolve in 1 L of distilled water to obtain a preparative solution, and adjust the pH value of the preparative solution to 8.3-8.6 with a nitric acid solution to prepare THE electrophoresis buffer.

The present invention also provides the application of the above-prepared THE electrophoresis buffer, and the THE electrophoresis buffer is used in electrophoresis experiments.

Compared with the prior art, the present invention has the following advantages:

Compared with the traditional electrophoresis buffer of sodium barbital barbiturate, the THE electrophoresis buffer prepared by the method of the invention has clearer and tidy zones, is easy to distinguish, has a high experimental success rate, and has a high probability of five zone results. NaCl effectively reduces the probability of electroosmosis, and the addition of NaCl increases the ionic strength of the buffer system, making it more suitable for electrophoresis experiments. The pH of the aqueous solution of Tris base is about 10.5. Add nitric acid reagent to adjust the pH value to the required value, and then the buffer solution with this pH value can be obtained. Tris base is easy to purchase as a common buffer solution. The nitric acid used is close to a trace amount, and disodium EDTA is added. , as a metal complexing agent, it is common and easy to buy, and it effectively complexes magnesium ions in serum.

In particular, THE buffer is not limited to an electrophoresis experiment, and experiments such as agarose gel electrophoresis and nucleic acid electrophoresis need to be studied.

The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

FIG. 1 is the experimental result of the THE electrophoresis buffer prepared in Example 2. FIG.

FIG. 2 is the experimental result of barbital-barbital sodium buffer in Example 2. FIG.

FIG. 3 is the experimental result of the THE1 electrophoresis buffer prepared in Comparative Example 1. FIG.

FIG. 4 is the experimental result of barbital-barbital sodium buffer in Comparative Example 1. FIG.

Detailed ways

Example 1

The electrophoresis buffer of this embodiment is prepared by adjusting the pH to 8.3-8.6 from the following raw materials: tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water.

The dosage ratio of the tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water is 10.800g: 0.930g: 1.317g: 1L; nitric acid solution is used to adjust the pH, and the mass fraction of the nitric acid solution is 50%.

The preparation method of the electrophoresis buffer of the present embodiment comprises the following steps:

The tris-base with a mass of 10.800g, disodium EDTA with a mass of 0.930g and NaCl with a mass of 1.317g were added into distilled water with a volume of 1L and fully dissolved to obtain a preparatory solution. The pH value of the preparative solution was adjusted with a nitric acid solution. Adjust to 8.3 to prepare THE running buffer.

The cellulose acetate film electrophoresis experiment was carried out using the electrophoresis buffer prepared in this example.

1. Test material:

Equipment: analytical balance, electrophoresis apparatus, electrophoresis tank, spotter, iron stand (with iron clip), acid burette (50ml), several beakers of different ranges, glass rod, pH meter;

2. Reagents:

(1) Fresh serum (rabbit serum, no coagulation during preparation);

(2) THE electrophoresis buffer prepared in this example;

(3) Dyeing solution: 0.5g of amino black 10B, dissolved in 40ml of distilled water, 50ml of methanol, 10ml of glacial acetic acid, fully mixed and stored in a reagent bottle;

(4) Rinse solution: mix 45ml of 95% ethanol, 5ml of glacial acetic acid and 50ml of distilled water;

(5) Acetate film (2x8cm).

3. Soak

Pour a small amount of THE electrophoresis buffer prepared in this example into the plate a, then identify the matte surfaces of the two cellulose acetate films, and make a mark with a pencil. A straight line perpendicular to the length of the membrane indicates the spotting position and marks a1, a2, then put the matte side down, soak the plate a in the electrophoresis buffer, let the film sink naturally, and soak for 30 minutes.

4. Spotting

The fully soaked film was removed from plate a, sandwiched between two layers of coarse filter paper to absorb excess buffer, and then the film was placed flat on a clean glass plate with the matte side up. Take a glass slide, suck fresh serum onto the glass slide with a 20ul pipette, tilt the glass slide to spread the serum evenly, then use a spotter to dab the serum on the surface, and then place a pencil mark on one end of the film. Spot upright gently and lift with it. This results in a thin strip of serum sample spotted on the film.

5. Electrophoresis

Place the matte surfaces of the two film strips a1 and a2 with samples, that is, the spotting surface, on the filter paper bridge of the electrophoresis tank downwards (use four layers of filter paper as bridges, and use buffer to completely wet the filter paper and drive out air bubbles, Make the filter paper close to the slot frame (the filter paper bridge), the film and the filter paper should be tightly attached, cover the cover, wait for 10 minutes to balance, and then power on, adjust the current intensity to 20mA, the voltage to 70v, power on for 50 minutes, and turn off the power.

6. Dyeing

After electrification, the film was taken out with toothless tweezers, directly immersed in a petri dish filled with amino black staining solution, and taken out after 5 minutes.

7. Rinse

Immerse the stained film in a petri dish filled with rinsing solution, and rinse 3 times repeatedly to remove the background color.

Repeat the above steps 10 times to obtain cellulose acetate films a1, a2, a3, a4, a5, a6, a7, a8, a9, a10, a11, a12, a13, a14, a15, a16, a17, a18, a19, a20, a total of 20.

8. Experimental results

Table 1 The number of bands that appear in the electrophoresis experiment of cellulose acetate film by the electrophoresis buffer prepared in this example

acetate film a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 The number of zones that appear 5 5 5 5 5 5 5 4 5 5 acetate film a11 a12 a13 a14 a15 a16 a17 a18 a19 a20 The number of zones that appear 5 4 5 5 5 5 5 5 5 5

It can be obtained from Table 1 that there are 20 results in total, of which the number of occurrences of five is 18, and the number of occurrences of four is 2.

The probability of the appearance of five cellulose acetate films is:

P=18/20*100%=90%

Therefore, it can be seen that the experimental success rate of the THE electrophoresis buffer prepared in this example in the serum protein acetate cellulose film is 100%, and the probability of five bands is 90%.

Example 2

The electrophoresis buffer of this embodiment is prepared by adjusting the pH to 8.3-8.6 from the following raw materials: tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water.

The dosage ratio of the tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water is 10.800g: 0.930g: 1.317g: 1L; nitric acid solution is used to adjust the pH, and the mass fraction of the nitric acid solution is 50%.

The preparation method of the electrophoresis buffer of the present embodiment comprises the following steps:

The mass of 10.800g of tris-alkali, the mass of 0.930g of disodium EDTA and the mass of 1.317g of NaCl were added to distilled water with a volume of 1L to obtain a preparatory solution, and the pH of the preparatory solution was adjusted to nitric acid solution. 8.6, prepare THE running buffer.

Using the electrophoresis buffer prepared in this example and the pH 8.6 barbital-barbital sodium buffer, the cellulose acetate film electrophoresis experiment was compared.

1. Test material:

Equipment: analytical balance, electrophoresis apparatus, electrophoresis tank, spotter, iron stand (with iron clip), acid burette (50ml), several beakers of different ranges, glass rod, pH meter;

2. Reagents:

(1) Fresh serum (rabbit serum, no coagulation during preparation);

(2) THE electrophoresis buffer prepared in this example; barbital-barbital sodium electrophoresis buffer (pH 8.6);

(3) Dyeing solution: 0.5g of amino black 10B, dissolved in 40ml of distilled water, 50ml of methanol, 10ml of glacial acetic acid, fully mixed and stored in a reagent bottle;

(4) Rinse solution: mix 45ml of 95% ethanol, 5ml of glacial acetic acid and 50ml of distilled water;

(5) Acetate film (2x8cm).

3. Soak

Pour a small amount of THE electrophoresis buffer prepared in this example into plate 1, then identify the matte surface of a cellulose acetate film, and mark it with a pencil. A straight line perpendicular to the length of the membrane indicates the spotting position and marks No. 1, then put the matte side down, soak the plate 1 in the electrophoresis buffer, let the membrane sink naturally, and soak for 30 minutes.

Pour a small amount of barbital-barbital sodium electrophoresis buffer into the plate 2, then identify the matte side of a cellulose acetate film, and mark it with a pencil. Draw a straight line perpendicular to the length of the membrane to indicate the spotting position and mark No. 2, then put the matte side down, soak the plate 2 in the electrophoresis buffer, let the membrane sink naturally, and soak for 30 minutes.

4. Spotting

The fully soaked film was taken out from plate 1 and plate 2, sandwiched between two layers of coarse filter paper to absorb excess buffer, and then placed the film with the matte side up on a clean glass plate. Take a glass slide, use a 20ul pipette to draw fresh serum onto the glass slide, tilt the slide glass to spread the serum evenly, and then use a spotter to dab the serum on the surface, and then place a pencil mark on one end of the film. Spot upright gently and lift with it. This results in a thin strip of serum sample spotted on the film.

5. Electrophoresis

Place the No. 1 and No. 2 film strips with samples on the matte surface, that is, the spotting surface, and place the filter paper bridge vertically downward on the filter paper bridge of the electrophoresis tank (use four layers of filter paper as the bridge, and use buffer to completely wet and drive off the filter paper. Air bubbles, make the filter paper close to the slot frame (the filter paper bridge), the film and the filter paper need to be close. Cover the lid, power on after 10 minutes of equilibration, adjust the current intensity to 20mA, the voltage to 70v, power on for 50 minutes, and turn off the power.

6. Dyeing

After electrification, the film was taken out with toothless tweezers, directly immersed in a petri dish filled with amino black staining solution, and taken out after 5 minutes.

7. Rinse

Immerse the stained film in a petri dish filled with rinsing solution, and rinse 3 times repeatedly to remove the background color.

Repeat the above steps 10 times to obtain cellulose acetate films b1, b2, b3, b4, b5, b6, b7, b8, b9, b10, b11, b12, b13, b14, b15, b16, b17, b18, b19, b20, a total of 20.

8. Experimental results

As shown in Figure 1, the experimental results of the THE electrophoresis buffer prepared in this example (No. 1): there are 5 clear bands on the film, starting from the positive end, followed by albumin, α1-globulin, α2- globulin, beta-globulin and gamma-globulin.

As shown in Figure 2, the experimental results of barbital-barbital sodium buffer solution: (No. 2): There can be four clear bands on the film, starting from the positive end, followed by albumin, α1-globulin, α2, β-globulin and γ-globulin.

Comparative Example 1

The electrophoresis buffer of this comparative example was prepared by adjusting the pH to 8.3-8.6 from the following raw materials: tris-hydroxymethylaminomethane (tris-base), disodium EDTA and distilled water.

The dosage ratio of the tris-hydroxymethylaminomethane (tris-base), disodium EDTA and distilled water is 10.800g:0.930g:1L; nitric acid solution is used for pH adjustment, and the mass fraction of the nitric acid solution is 50%.

The preparation method of the electrophoresis buffer of this comparative example includes the following steps:

Add 10.800 g of tris-base and 0.930 g of disodium EDTA into distilled water with a volume of 1 L to obtain a preparative solution, and adjust the pH of the preparative solution to 8.6 with nitric acid solution to prepare THE1 electrophoresis buffer liquid.

Using the THE1 electrophoresis buffer prepared in this comparative example and the pH8.6 barbital-barbital sodium buffer, the cellulose acetate film electrophoresis experiment was compared.

1. Test material:

Equipment: analytical balance, electrophoresis apparatus, electrophoresis tank, spotter, iron stand (with iron clip), acid burette (50ml), several beakers of different ranges, glass rod, pH meter;

2. Reagents:

(1) Fresh serum (rabbit serum, no coagulation during preparation);

(2) THE1 electrophoresis buffer prepared in this comparative example; barbital-barbital sodium electrophoresis buffer (pH 8.6);

(3) Dyeing solution: 0.5g of amino black 10B, dissolved in 40ml of distilled water, 50ml of methanol, 10ml of glacial acetic acid, fully mixed and stored in a reagent bottle;

(4) Rinse solution: mix 45ml of 95% ethanol, 5ml of glacial acetic acid and 50ml of distilled water;

(5) Acetate film (2x8cm).

3. Soak

Pour a small amount of THE1 electrophoresis buffer prepared in this comparative example into plate 3, then identify the matte surface of a cellulose acetate film, and mark it with a pencil. A straight line perpendicular to the length of the membrane indicates the spotting position and marks No. 3, then put the matte side down, soak the plate 3 in the electrophoresis buffer, let the membrane sink naturally, and soak for 30 minutes.

Pour a small amount of barbital-barbital sodium electrophoresis buffer into the plate 4, then identify the matte side of a cellulose acetate film, and make a mark with a pencil. Draw a straight line perpendicular to the length of the membrane to indicate the spotting position and mark No. 4, then put the matte side down, soak the plate 4 in the electrophoresis buffer, let the membrane sink naturally, and soak for 30 minutes.

4. Spotting

The fully soaked film was taken out from plate 3 and plate 4, sandwiched between two layers of coarse filter paper to absorb excess buffer, and then placed the film with the matte side up on a clean glass plate. Take a glass slide, suck fresh serum onto the glass slide with a 20ul pipette, tilt the glass slide to spread the serum evenly, then use a spotter to dab the serum on the surface, and then place a pencil mark on one end of the film. Spot upright gently and lift with it. This results in a thin strip of serum sample spotted on the film.

5. Electrophoresis

Place the No. 3 and No. 4 film strips with samples on the matte surface, i.e., the spotting surface, with the vertical filter paper bridge facing downwards on the filter paper bridge of the electrophoresis tank (use four layers of filter paper as bridges, and use buffer to completely wet the filter paper and drive it off) Air bubbles, make the filter paper close to the slot frame (the filter paper bridge), the film and the filter paper need to be close. Cover the lid, power on after 10 minutes of equilibration, adjust the current intensity to 20mA, the voltage to 70v, power on for 50 minutes, and turn off the power.

6. Dyeing

After electrification, the film was taken out with toothless tweezers, directly immersed in a petri dish filled with amino black staining solution, and taken out after 5 minutes.

7. Rinse

Immerse the stained film in a petri dish filled with rinsing solution, and rinse 3 times repeatedly to remove the background color.

Repeat the above steps 10 times to obtain cellulose acetate films c1, c2, c3, c4, c5, c6, c7, c8, c9, c10, c11, c12, c13, c14, c15, c16, c17, c18, c19, c20, a total of 20.

8. Experimental results

As shown in Figure 3, the experimental results of the THE1 electrophoresis buffer prepared in this comparative example (No. 3): there can be five clear bands on the film, starting from the positive end, followed by albumin, α1-globulin, α2- globulin, beta-globulin and gamma-globulin.

As shown in Figure 4, the experimental results of barbital-barbital sodium buffer solution: (No. 4): There can be four clear bands on the film, starting from the positive end, followed by albumin, α1-globulin, α2, β-globulin and γ-globulin.

Based on the above, it can be found that the experimental results of the THE1 electrophoresis buffer prepared in Example 2 are better than the experimental results of the THE1 electrophoresis buffer and the barbital-barbital sodium buffer prepared in Comparative Example 1. The THE1 electrophoresis buffer prepared in Comparative Example 1 NaCl was not added to the buffer, but the electrophoresis buffer prepared in Example 2 was added with NaCl, which effectively reduced the probability of electroosmosis. At the same time, the addition of NaCl increased the ionic strength of the buffer system and was more suitable for electrophoresis experiments.

Example 3

The electrophoresis buffer of this embodiment is prepared by adjusting the pH to 8.3-8.6 from the following raw materials: tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water.

The dosage ratio of the tris-hydroxymethylaminomethane (tris-base), disodium EDTA, NaCl and distilled water is 10.800g: 0.930g: 1.317g: 1L; nitric acid solution is used to adjust the pH, and the mass fraction of the nitric acid solution is 50%.

The preparation method of the electrophoresis buffer of the present embodiment comprises the following steps:

The tris-base with a mass of 10.800g, disodium EDTA with a mass of 0.930g and NaCl with a mass of 1.317g were added into distilled water with a volume of 1L and fully dissolved to obtain a preparatory solution. The pH value of the preparative solution was adjusted with a nitric acid solution. Adjust to 8.4 to prepare THE running buffer.

The above descriptions are only preferred embodiments of the present invention, and do not limit the present invention in any way. Any simple modifications, changes and equivalent changes made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solutions of the present invention.

Claims (4)

1. An electrophoresis buffer solution is characterized by being prepared by adjusting the pH value to 8.3-8.6 by the following raw materials: tris, disodium EDTA, NaCl and distilled water.

2. The running buffer of claim 1, wherein the tris, disodium EDTA, NaCl, and distilled water are used in a ratio of 10.800 g: 0.930 g: 1.317 g: 1L of the compound.

3. An electrophoresis buffer according to claim 1 or 2 wherein the pH is adjusted using a nitric acid solution.

4. An electrophoresis buffer according to claim 3 wherein the nitric acid solution is present in an amount of 50% by weight.

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