Colorful dyeing-free SDS-PAGE polyacrylamide gel

Abstract

The invention discloses SDS-PAGE polyacrylamide gel prepared by a one-step method, the SDS-PAGE polyacrylamide gel comprises a separation gel and a concentration gel, the separation gel comprises the following components: a Tris-HCl buffer solution, acrylamide, N, N '-methylene bisacrylamide, a trihalide compound, lauryl sodium sulfate, an ammonium persulfate solution, a glycerin aqueous solution, tetramethylethylenediamine and water; the concentrated gel is prepared from the following components: a Tris-HCl buffer solution, acrylamide, N, N '-methylene bisacrylamide, lauryl sodium sulfate, an ammonium persulfate solution, tetramethylethylenediamine and ultrapure water. The polyacrylamide gel provided by the invention can be used for preparing gel by a one-step method, so that a sample loading hole is clearer and more visible, and a clear protein band can be presented by irradiating for 60-90 seconds under 300 nm ultraviolet after electrophoresis.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation in the field of bioelectrophoresis, and in particular relates to a color stain-free SDS-PAGE polyacrylamide gel. Background Art

[0002] Polyacrylamide gel electrophoresis, also known as PAGE (polyacrylamide gel electrophoresis), is an electrophoresis technique that uses polyacrylamide gel as a supporting medium and is often used to separate mixed protein samples. It is divided into two forms: native-PAGE and denaturing polyacrylamide gel electrophoresis (SDS-PAGE). It is mainly a three-dimensional network structure formed by acrylamide monomer and cross-linker N,N'-methylenebisacrylamide under the catalysis of ammonium persulfate. In SDS-PAGE, SDS carries a large amount of negative charge and combines with protein to form an SDS-protein complex. The negative charge carried by the complex far exceeds the charge carried by the protein itself, which masks the difference between the charges of the protein itself. At this time, the SDS-protein complex presents an elliptical rod shape, which is only related to the molecular weight of the protein subunit, so that during electrophoresis, the protein can be separated only according to the molecular weight of the protein subunit. The SDS-PAGE gel commonly used in laboratories is a discontinuous polyacrylamide gel system, which is divided into two components: a concentration gel and a separation gel. During electrophoresis, the concentration gel is located in the upper layer of the vertical electrophoresis system and has a concentration effect. The separation gel is located in the lower layer. After the protein mixed sample enters the separation gel, it will be separated according to the molecular weight of the protein itself. This technology is the most effective means of protein sample separation and detection currently used in laboratories.

[0003] The preparation of ordinary protein separation gel needs to be divided into two steps: separation gel and concentration gel. After the separation gel is poured, it is necessary to wait for 30 minutes for it to solidify before preparing the concentration gel. The whole preparation process is time-consuming; the concentration gel loading hole of ordinary protein separation gel is transparent and small, which is difficult for the operator to see clearly. For novice operators, finding the loading hole is even more challenging; and after the electrophoresis, it needs to be stained with Coomassie Brilliant Blue R-250 or G-250, and then repeatedly eluted with a decolorizing solution containing organic matter such as methanol and acetic acid to observe the bands. Staining and repeated decolorization take a long time, about 2-4 hours, and methanol and acetic acid are both hazardous chemicals, which are difficult to purchase. The methanol and acetic acid mixed solution that the experimenter needs to repeatedly contact is not only irritating but also toxic. While seriously damaging the health of the experimenter, the harm of the generated organic waste to the environment cannot be underestimated. Summary of the invention

[0004] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a color-free SDS-PAGE polyacrylamide gel.

[0005] The technical solution adopted by the present invention is: In a first aspect, the present invention provides a one-step prepared SDS-PAGE polyacrylamide gel, comprising a separation gel and a concentration gel, wherein the separation gel comprises the following components: Tris-HCl buffer, acrylamide, N,N'-methylenebisacrylamide, a trihalogen compound, sodium dodecyl sulfate, ammonium persulfate solution, glycerol aqueous solution, tetramethylethylenediamine and water; The concentrated gel comprises the following components: Tris-HCl buffer, acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate, ammonium persulfate solution, tetramethylethylenediamine and ultrapure water.

[0006] In some examples, the volume content ratio of each component in the separation gel is as follows: 1.2-5.0 parts of Tris-HCl buffer, 2.0-6.0 parts of acrylamide mixed solution, 0.025-0.1 parts of trihalogen compound, 0.05-0.2 parts of sodium dodecyl sulfate solution, 0.5-0.2 parts of ammonium persulfate solution, 0.5-2.0 parts of glycerol, 0.004-0.008 parts of tetramethylethylenediamine, and 1.0-10.0 parts of water.

[0007] In some examples, the concentration of the glycerol aqueous solution is 0.095-0.105 mL / mL.

[0008] In some examples, it is characterized in that, in the separation gel, the Tris concentration in the Tris-HCl buffer is 1.45-1.55 mol / L, and the pH is adjusted to 8.7-8.9 with hydrochloric acid.

[0009] In some examples, the trihalogen compound is selected from at least one of chloroform, trichloroacetic acid and trichloroethanol.

[0010] In some examples, an appropriate amount of acid dye is added to the stacking gel. The acid dye is used to make the sample loading wells of the stacking gel clearly visible, so that the sample loading wells can be displayed.

[0011] In some examples, the volume content ratio of each component in the concentrated gel is as follows: 0.2-1.0 parts of Tris-HCl buffer, 1.0-5.0 parts of acrylamide mixed solution, 0.01-0.1 parts of sodium dodecyl sulfate solution, 0.01-0.1 parts of ammonium persulfate solution, 0.004-0.006 parts of tetramethylethylenediamine, 0.02-0.06 parts of acid dye solution, and 1.0-10.0 parts of ultrapure water.

[0012] In some examples, the acid dye is selected from at least one of lemon yellow, brilliant blue FCF, or new carmine.

[0013] In some examples, in the stacking gel, the Tris concentration in the Tris-HCl buffer is 0.95-1.05 mol / L, and the pH is adjusted to 6.7-6.9 with hydrochloric acid.

[0014] The above features can be combined arbitrarily as long as they do not conflict with each other.

[0015] In a second aspect, the present invention provides a method for preparing a stain-free SDS-PAGE polyacrylamide gel, comprising the following steps: Adding the prepared separation gel solution into a vertical glass plate to form a separation gel, wherein the separation gel is as described in the first aspect; Without waiting for the separation gel to solidify, the prepared concentrated gel solution is added to the vertical gel-making glass plate, a comb is inserted, and after the concentrated gel solidifies above the separation gel, a stain-free SDS-PAGE polyacrylamide gel is obtained, and the concentrated gel is as described in the first aspect.

[0016] The beneficial effects of the present invention are: The SDS-PAGE polyacrylamide gel of some examples of the present invention improves the separation gel formula so that the concentrated gel can be directly poured before the separation gel solidifies, thereby overcoming the problem that the traditional protein gel needs to stand for half an hour after the separation gel is prepared to solidify before the concentrated gel can be poured, realizing a one-step gel preparation, significantly simplifying the gel preparation process and shortening the gel preparation time.

[0017] In some embodiments of the SDS-PAGE polyacrylamide gel of the present invention, acid dye is added to the concentrated gel, so that the sample loading wells are more clearly visible, thereby facilitating the sample loading operation.

[0018] The SDS-PAGE polyacrylamide gel of some examples of the present invention utilizes trihalide to combine with tryptophan, and under the light activation of 300 nm ultraviolet wavelength, the protein band containing tryptophan can be made to emit light in the gel, and after electrophoresis, it can be realized that clear protein bands can be presented by irradiating under 300 nm ultraviolet for 60-90 s. The problem that the traditional SDS-PAGE electrophoresis needs Coomassie brilliant blue staining for more than 30 minutes, and that organic reagents such as methanol and acetic acid are required for repeated decolorization after staining is overcome. The influence of Coomassie brilliant blue staining of different manufacturers and the decolorization degree of decolorization solution on the detection sensitivity is overcome, and the health hazards to the experimenters caused by the use of toxic and irritating organic substances such as methanol and acetic acid are avoided.

[0019] In some embodiments of the polyacrylamide gel of the present invention, color stain-free imaging during concentration degelation can eliminate the inherent problem of housekeeping proteins as a protein blot loading control, thereby being able to obtain truly quantitative protein blot data by normalizing the bands to the total protein in each lane. The color stain-free SDS-PAGE polyacrylamide gel of the present invention does not use special buffers or reagents and is compatible with the commonly used and inexpensive Tris-Glycine electrophoresis system. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a comparison of the photographs of Example 1 (right) and Example 4 (left) after the concentrated gel was solidified.

[0021] Figure 2 It is a comparison diagram of the SDS-PAGE polyacrylamide gel after solidification of Example 2 (left) and Comparative Example 3 (right). DETAILED DESCRIPTION

[0022] The present invention will be described in detail below with reference to specific embodiments.

[0023] Example 1: Preparation of 10% colored SDS-PAGE polyacrylamide gel The specific preparation method is as follows: 1) Accurately weigh 18.171 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 8.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 2) Accurately weigh 29 g acrylamide and 1 g N,N'-methylenebisacrylamide, dissolve them in 70 mL ultrapure water, stir until fully mixed, then make up to 100 mL with ultrapure water, and filter to remove impurities; 3) Accurately weigh 1 g of ammonium persulfate solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 4) Accurately measure 1 mL of glycerol and dissolve it in 8 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 5) Pipette 2.5 mL 1.5 mol / L Tris-HCl (pH=8.8) solution, 3.33 mL 30% polyacrylamide solution, 50 μL trihalogen compound mixture, 100 μL 10% ammonium persulfate solution, 100 μL 10% sodium dodecyl sulfate solution, 1 mL 10% glycerol solution and 4 μL tetramethylethylenediamine into a centrifuge tube with a scale line, then add ultrapure water to make up to 10 mL, invert and mix thoroughly to prepare a separation gel solution; 6) Add the separation gel solution obtained in step 5 to a 1.0 mm vertical glass plate to prepare a separation gel; 7) Accurately weigh 12.114 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 6.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 8) Accurately weigh 0.2 g of acid dye solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, make up to 10 mL with ultrapure water, and filter to remove impurities. 9) Pipette 0.63 mL of 1.0 mol / L Tris-HCl (pH=6.8) solution, 0.83 mL of 30% polyacrylamide solution, 50 μL of 10% sodium dodecyl sulfate, 50 μL of 10% ammonium persulfate solution, 40 μL of 2% acid dye solution and 5 μL of tetramethylethylenediamine into a centrifuge tube with a scale line, then add ultrapure water to make up to 5 mL, invert and mix thoroughly to prepare a stacking gel solution; 10) Add the concentrated gel solution prepared in step 9, then insert the comb and let it stand for 20-30 minutes to wait for the gel to solidify and form a polyacrylamide gel.

[0024] Example 2: Preparation of 12% Colored SDS-PAGE Polyacrylamide Gel The specific preparation method is as follows: 1) Accurately weigh 18.171 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 8.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 2) Accurately weigh 29 g acrylamide and 1 g N,N'-methylenebisacrylamide, dissolve them in 70 mL ultrapure water, stir until fully mixed, then make up to 100 mL with ultrapure water, and filter to remove impurities; 3) Accurately weigh 1 g of ammonium persulfate solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 4) Accurately measure 1 mL of glycerol and dissolve it in 8 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 5) Pipette 2.5 mL 1.5 mol / L Tris-HCl (pH=8.8) solution, 4 mL 30% polyacrylamide solution, 50 μL trihalogen compound mixture, 100 μL 10% SDS solution, 100 μL 10% ammonium persulfate solution, 1 mL 10% glycerol solution and 4 μL tetramethylethylenediamine into a centrifuge tube with a scale line, then add ultrapure water to make up to 10 mL, invert and mix thoroughly to prepare a separation gel solution; 6) Add the separation gel solution obtained in step 5 to a 1.0 mm vertical glass plate, add an appropriate amount of ultrapure water or isopropanol to press the line, and let it stand for about 15-30 minutes to solidify the separation gel to form a separation gel; 7) Accurately weigh 12.114 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 6.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 8) Accurately weigh 0.2 g of acid dye solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, make up to 10 mL with ultrapure water, and filter to remove impurities. 9) Pipette 0.63 mL of 1.0 mol / L Tris-HCl (pH=6.8) solution, 0.83 mL of 30% polyacrylamide solution, 50 μL of 10% sodium dodecyl sulfate, 50 μL of 10% ammonium persulfate solution, 40 μL of 2% acid dye solution and 5 μL of tetramethylethylenediamine into a centrifuge tube with scale lines, then add ultrapure water to make up to 5 mL, invert and mix thoroughly to prepare a stacking gel solution; 10) Add the concentrated gel solution prepared in step 9, then insert the comb and let it stand for 15-30 minutes to wait for the gel to solidify and form a polyacrylamide gel.

[0025] Example 3: Preparation of 15% colored non-staining SDS-PAGE polyacrylamide gel The specific preparation method is as follows: 1) Accurately weigh 18.171 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 8.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 2) Accurately weigh 29 g of acrylamide and 1 g of N,N'-methylenebisacrylamide, dissolve them in 70 mL of ultrapure water, stir until fully mixed, then make up to 100 mL with ultrapure water, and filter to remove impurities; 3) Accurately weigh 1 g of ammonium persulfate solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 4) Accurately measure 1 mL of glycerol and dissolve it in 8 mL of ultrapure water. Stir until fully mixed, then make up to 10 mL with ultrapure water and filter to remove impurities. 5) Pipette 2.5 mL of 1.5 mol / L Tris-HCl (pH=8.8) solution, 5 mL of 30% polyacrylamide solution, 50 μL of trihalogen compound mixture, 100 μL of 10% sodium dodecyl sulfate, 100 μL of 10% ammonium persulfate solution, 1 mL of 10% glycerol solution and 4 μL of tetramethylethylenediamine into a centrifuge tube with a scale line, then add ultrapure water to make up to 10 mL, invert and mix thoroughly to prepare a separation gel solution; 6) Add the separation gel solution obtained in step 5 to a 1.0 mm vertical glass plate, add an appropriate amount of ultrapure water or isopropanol to press the line, and let it stand for about 15-30 minutes to solidify the separation gel to form a separation gel; 7) Accurately weigh 12.114 g of Tris solid powder and dissolve it in 80 mL of ultrapure water. Stir until fully mixed. Adjust the pH to 6.8 with concentrated hydrochloric acid. Then make up to 100 mL with ultrapure water and filter to remove impurities. 8) Accurately weigh 0.2 g of acid dye solid powder and dissolve it in 9 mL of ultrapure water. Stir until fully mixed, make up to 10 mL with ultrapure water, and filter to remove impurities. 9) Pipette 0.63 mL of 1.0 mol / L Tris-HCl (pH=6.8) solution, 0.83 mL of 30% polyacrylamide solution, 50 μL of 10% sodium dodecyl sulfate, 50 μL of 10% ammonium persulfate solution, 40 μL of 2% acid dye solution and 5 μL of tetramethylethylenediamine into a centrifuge tube with a scale line, then add ultrapure water to make up to 5 mL, invert and mix thoroughly to prepare a stacking gel solution; 10) Add the concentrated gel solution prepared in step 9, then insert the comb and let it stand for 15-30 minutes to wait for the gel to solidify and form a polyacrylamide gel.

[0026] Embodiment 4: The difference from Example 1 is that no acid dye is added to the concentrated gel.

[0027] Comparative Example 1 The difference from Example 1 is that no glycerol and trihalogen compounds are added to the separation gel, that is, ordinary SDS-PAGE polyacrylamide gel.

[0028] Comparative Example 2 The difference from Example 1 is that no glycerol is added to the separation gel, and the color-free SDS-PAGE polyacrylamide gel is used.

[0029] Comparative Example 3 The difference from Example 1 is that glycerol is not added to the separation gel, and the concentration gel is directly poured in after the separation gel is poured but before it solidifies.

[0030] Protein electrophoresis The electrophoresis test was performed using the colored non-staining SDS-PAGE polyacrylamide gels of different concentrations prepared in the above examples and comparative examples, and the specific operation was as follows: Different bacteria were lysed and added to loading buffer for boiling sample preparation, and then the samples were added to the above-mentioned gel loading wells respectively. The electrophoresis buffer used was the Tris-Glycine electrophoresis system, the voltage was set at 120 V, and a protein marker was added as a measurement standard. The electrophoresis time was set to 45 min. After the electrophoresis was completed, the gel block was taken out and irradiated under 300nm ultraviolet light for 60-90 s for experimental observation.

[0031] The specific steps to verify the efficiency of protein transfer from gel to membrane are as follows: 1) Soak the nitrocellulose membrane and foam in transfer buffer for 30 min; 2) Place the foam, filter paper, nitrocellulose membrane, and protein gel after electrophoresis in a "sandwich" format and place in the transfer tank; 3) Electrophoresis at 50 V for one hour and thirty minutes; 4) Take out the gel block and expose it to 300 nm UV light for 60-90 s to observe the transfer efficiency.

[0032] The comparison results are shown in Table 1.

[0033] Table 1 Comparison of the characteristics of ordinary, stain-free and one-step stain-free SDS-PAGE polyacrylamide gels

[0034] Figure 1 The following are photos of the concentrated gel after solidification in Example 1 (right) and Example 4 (left). It can be seen that without adding acid dye, the sample loading hole is not obvious, which is not conducive to accurate sample loading. There is a clear boundary line between the concentrated gel and the separation gel in Example 1, indicating that the separation gel and the concentrated gel are not miscible.

[0035] Figure 2 The SDS-PAGE polyacrylamide gels after solidification of Example 2 (left) and Comparative Example 3 (right) show that the addition of glycerol to the separation gel can surprisingly prevent the unsolidified separation gel and the concentrated gel from mixing. The separation gel without glycerol will mix with the concentrated gel before solidification.

[0036] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A one-step prepared SDS-PAGE polyacrylamide gel, comprising a separation gel and a concentration gel, characterized in that: The separation gel comprises the following components: Tris-HCl buffer, acrylamide, N,N'-methylenebisacrylamide, trihalogen compound, sodium dodecyl sulfate, ammonium persulfate solution, glycerol aqueous solution, tetramethylethylenediamine and water; The concentrated gel comprises the following components: Tris-HCl buffer, acrylamide, N,N'-methylenebisacrylamide, sodium dodecyl sulfate, ammonium persulfate solution, tetramethylethylenediamine and ultrapure water.

2. The SDS-PAGE polyacrylamide gel according to claim 1, characterized in that The volume content ratio of each component in the separation gel is as follows: 1.2-5.0 parts of Tris-HCl buffer, 2.0-6.0 parts of acrylamide mixed solution, 0.025-0.1 parts of trihalogen compound, 0.05-0.2 parts of sodium dodecyl sulfate solution, 0.5-0.2 parts of ammonium persulfate solution, 0.5-2.0 parts of glycerol, 0.004-0.008 parts of tetramethylethylenediamine, and 1.0-10.0 parts of water.

3. The SDS-PAGE polyacrylamide gel according to claim 1, characterized in that The concentration of the glycerol aqueous solution is 0.095-0.105 mL / mL.

4. The SDS-PAGE polyacrylamide gel according to claim 1, characterized in that In the separation gel, the concentration of Tris in the Tris-HCl buffer is 1.45-1.55 mol / L, and the pH is adjusted to 8.7-8.9 with hydrochloric acid.

5. The SDS-PAGE polyacrylamide gel according to any one of claims 1 to 4, characterized in that: The trihalogen compound is selected from at least one of chloroform, trichloroacetic acid and trichloroethanol.

6. The SDS-PAGE polyacrylamide gel according to claim 1, characterized in that A proper amount of acid dye is also added to the concentrated gel.

7. The SDS-PAGE polyacrylamide gel according to claim 6, characterized in that The volume content ratio of each component in the concentrated gel is as follows: 0.2-1.0 parts of Tris-HCl buffer, 1.0-5.0 parts of acrylamide mixed solution, 0.01-0.1 parts of sodium dodecyl sulfate solution, 0.01-0.1 parts of ammonium persulfate solution, 0.004-0.006 parts of tetramethylethylenediamine, 0.02-0.06 parts of acid dye solution, and 1.0-10.0 parts of ultrapure water.

8. The SDS-PAGE polyacrylamide gel according to claim 6, characterized in that The acid dye is selected from at least one of lemon yellow, brilliant blue FCF or new carmine.

9. The SDS-PAGE polyacrylamide gel according to any one of claims 1 to 4 and 6 to 8, characterized in that In the concentrated gel, the Tris concentration in the Tris-HCl buffer is 0.95-1.05 mol / L, and the pH is adjusted to 6.7-6.9 with hydrochloric acid.

10. A method for preparing a stain-free SDS-PAGE polyacrylamide gel, characterized in that: The steps include: Adding the prepared separation gel solution to a vertical glass plate to form a separation gel, wherein the separation gel is as described in any one of claims 1 to 5; Without waiting for the separation gel to solidify, the prepared concentrated gel solution is added to a vertical gel-making glass plate, a comb is inserted, and after the concentrated gel solidifies above the separation gel, a stain-free SDS-PAGE polyacrylamide gel is obtained, wherein the concentrated gel is as described in any one of claims 1, 6 to 9.

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