Protein Technology Topics: Preparation of Protein Gel Electrophoresis Gel

【Materials and Reagents】

(1) 30% acrylamide: weigh 29g acrylamide, 1g N, N-methylene bis acrylamide and warm deionized water 60ml, heat to 37 ° C to dissolve, add water to a final volume of 100ml, filter That is, formulated into a 30% (w/v) acrylamide storage solution. Acrylamide and bisacrylamide are slowly converted to acrylic acid and diacrylic acid during storage. This deamination reaction is photocatalytic or base catalyzed, so the pH of the solution does not exceed 7.0 and should be stored in a brown bottle at 4 ° C.

(2) 10% sodium dodecyl sulfate (SDS): Weigh 10g SDS plus deionized water 90ml and heat to 68 ° C, add a few drops of concentrated hydrochloric acid to adjust to pH 7.2 and add water to 100ml, which is 10% (w / v ) SDS.

(3) Concentrated gel buffer (1 mol/L Tris-HCl pH 6.8): 12.12 g Tris was dissolved in 80 ml of deionized water, adjusted to pH 6.8 with concentrated hydrochloric acid, and deionized water was added to 100 ml. Store at 4 ° C.

(4) Separation gel buffer (1.5 mol/L Tris-HCl pH 8.8): 18.16 g Tris was dissolved in 80 ml of deionized water, adjusted to pH 8.8 with concentrated hydrochloric acid, and deionized water was added to 100 ml. Store at 4 ° C.

(5) 10% persulfate amine (AP): Persulfate amine provides the free radicals necessary for the polymerization of acrylamide and bisacrylamide. A small amount of 10% (W/V) stock solution can be prepared with deionized water and stored at 4 ° C. . Since ammonium persulfate will slowly decompose, it should be freshly prepared every other week.

(6) TEMED (N, N, N, N - tetramethylethylenediamine) TEMED accelerates the polymerization of acrylamide and bisacrylamide by catalyzing the formation of free radicals by ammonium persulfate, since TEMED can only be used as a free base. The effect is that the polymerization reaction is inhibited when the pH is low.

(7) Tris-glycine electrophoresis buffer: Weigh 15.1 g of Tris and 94 g of glycine, dissolve in 900 ml of deionized water, then add 50 ml of 10% (w/v) SDS, then add deionized water to 1000 ml. Into 5? stock solution. It was diluted 5 times when used, and the final concentration of Tris was 25 mmol/L, glycine was 250 mmol/L, 0.1% SDS, and the buffer pH was (8.3).

(8) Polyacrylamide gel electrophoresis tank and electrophoresis apparatus

(9) Sampler, tip, etc.

[Operation method]

(1) Install the glass plate according to the instructions of the vertical electrophoresis tank, determine the concentration and volume of the separation gel to be prepared, and prepare according to the ingredients listed in the “Preparation of Tris-Glycine SDS Polyacrylamide Gel Electrophoresis Separation Gel Solution” (see appendix at the end of the book). Separating glue required;

(2) Quickly inject the separation glue into the gap between the two glass plates, leaving the space required for pouring the laminated glue (add 1 cm to the tooth length of the comb, carefully cover the separation rubber with 0.1% SDS with a dropper (when propylene When the amide concentration is ≤ 8% or isobutanol or water (when the acrylamide concentration is ≥ 10%), the coating layer prevents oxygen from diffusing into the gel and inhibits the polymerization reaction. The gel is placed vertically at room temperature;

(3) After the separation gel is completely polymerized, the cover liquid is poured out, and the top of the gel is washed several times with deionized water to remove unpolymerized acrylamide, and the liquid on the gel is excluded as much as possible;

(4) Determine the volume of the concentrated gel to be prepared, and prepare the required concentrated gel according to the “Preparation of Tris-Glycine SDS Polyacrylamide Gel Electrophoresis Gel Concentrate” (see appendix at the end of the book), and then directly inject the concentrated gel into the separation gel. Immediately, insert a clean matching comb to avoid air bubbles, and then add a concentrated glue solution to fill the gap between the combs. After the concentrated gel is polymerized, the comb is pulled out to form a sample loading hole;

(5) Dilute the Tris-Glycine running buffer stock solution by 5 times with deionized water, pour into the electrophoresis tank, and fill the well. At this time, the bubbles in the well can be removed by running buffer.

High Content Monomer

Plant extraction process