SDS-polyacrylamide gel electrophoresis (PAGE)

【Fundamental】

After a mixed protein sample was subjected to polyacrylamide gel electrophoresis, the components were separated due to different electrophoretic mobility. This difference is primarily related to the molecular weight and the net charge and shape of the protein molecule itself. When the electrophoresis system contains a certain concentration of SDS, the electrophoretic mobility depends only on the molecular weight of the protein (other factors are negligible), so that the molecular weight of the protein can be directly calculated from the electrophoretic mobility.

SDS (sodium lauryl sulfate) is an anionic detergent that binds to proteins, destroys non-covalent bonds between proteins, molecules, and other molecules, denatures proteins and alters the original. There is a spatial conformation. When a strong reducing agent (such as mercaptoethanol) is present, the disulfide bond in the protein molecule is completely reduced, and the total amount of SDS is 3 to 10 times the amount of protein, and the unit concentration of SDS is more than 1 mol/L. The combination of the two is quantitative, and approximately 1.4 grams of SDS can be combined per gram of protein. Once a protein molecule has been combined with a certain amount of SDS anion, the amount of negative charge is far greater than its original charge. Thereby eliminating the difference in charge between different kinds of proteins, and because the larger the molecular weight, the more SDS is bound by the protein, the more negatively charged, which makes the charge density of each protein-SDS complex tend to be uniform; The SDS complexes of proteins are also similar in shape and are all oblong. Therefore, during electrophoresis, the mobility depends on the size of the protein-SDS complex, or it depends on the molecular weight of the protein.

It is known from experience that when the molecular weight of the protein is between 17 000 and 165,000. The electrophoretic mobility of the protein-SDS complex is linear with the logarithm of the molecular weight of the protein:

lgMW=lgK-bm

Where MW is the molecular weight of the protein, m is the relative mobility, K is a constant, and b is the slope. A standard curve is obtained by plotting the electrophoretic mobility of a standard protein of known molecular weight in a SDS-polyacrylamide gel versus the logarithm of the molecular weight. As long as the electrophoretic mobility of an unknown molecular weight protein under the same conditions is measured, its molecular weight can be determined from a standard curve.

【equipment】

Electrophoresis apparatus

2. Vertical plate electrophoresis tank

3. Microsampler

4. Nipple pipette

5.50ml small beaker

【Reagents】

1. Preparation of stock solution

(1) 30% acrylamide solution: 29.2 g of acrylamide, 0.8 g of methylidene bisacrylamide, water was added to 100 ml, and the brown bottle was stored at 4 ° C.

(2) 1.5 mmol/LTris-Cl separation gel buffer, pH 8.8 (4×): Weigh 18.15 g of Tris, adjust the pH to 8.8 with 1N HCl, add water to 100 ml, and store at 4 °C.

(3) 1.0 mmol/L Tris-Cl concentrated gel buffer, pH 6.8 (4×): Weigh 5.98 g of Tris, adjust the pH to 6.8 with 1N HCl, add water to 100 ml, and store at 4 °C.

(4) Electrode buffer (pH 8.3): 14.40 g of glycine, 3.00 g of Tris, 10 ml of 10% SDS, water to 1 L, and stored at 4 ° C.

(5) 10% SDS: Take 10 g of SDS, add water to 100 ml, completely dissolve and store at room temperature.

(6) 10% ammonium persulfate solution (AP): ready for use before use, or after -20 °C.

(7) Staining solution (0.25% Coomassie Brilliant Blue R-250, 50% methanol, 7% acetic acid): Coomassie Brilliant Blue R-250 2.5g, methanol 500ml, 70ml glacial acetic acid, add water to a total volume of 1000ml (methanol can be used without water) Ethanol instead).

(8) Decolorization solution (30% methanol, 7% acetic acid): methanol 300 ml, glacial acetic acid 70 ml, and water is added to 1000 ml (anhydrous ethanol can also be used instead of methanol).

(9) Sample buffer (2×): H2O 2.4 ml, concentrated gel buffer 1.0 ml, glycerol 0.8 ml, 10% SDS

3.2 ml, 0.4 ml of 2-thioethanol. 0.025% (w/v) bromophenol blue 0.2 ml.

(10) TEMED

2. Preparation of working fluid

Shanghai Chuangsai Technology has excellent performance, interleukin cytokines, fetal bovine serum, electrophoresis equipment scientific instruments, raw material drug standards, chemical reagents, cell culture consumables, Shanghai Chuangsai, mass products special promotions, welcome to inquire!

Filing Cabinet

Filing Cabinet,Cabinet For Living Room Furniture,Wooden Shoe Cabinet,File Cabinet With Wheels

Longsheng Office Furniture Co.,Ltd , https://www.longshengfurniture.com