Determining The Relative Molecular Mass of Proteins Using SDS-PAGE

The determination of the relative molecular mass of proteins is of great significance in theory and practice. When a protein is electrophoresed in a polyacrylamide gel, its mobility depends on factors such as its net charge and the size and shape of the molecule. In 1967, Shapiro et al. discovered that by adding excess anionic detergents sodium dodecyl sulfate (SDS) and mercaptoethanol to the sample, the protein was denatured and depolymerized, and SDS combined with the protein to form a strongly negatively charged complex, masking the original charge difference between proteins. The combination of SDS and protein molecules not only makes the protein molecules carry a large number of negative charges, but also changes the shape of the protein molecules into short rods, thereby eliminating the original charge differences and molecular shape differences between protein molecules. Therefore, the mobility of proteins in SDS-PAGE mainly depends on their molecular size, which makes SDS-PAGE electrophoresis have the characteristics of high resolution and good repeatability.

Principle

Add a certain amount of SDS to the polyacrylamide gel system. SDS is an anionic surfactant. When added to the electrophoresis system, it can open the hydrogen bonds and hydrophobic bonds of proteins and bind to protein molecules, making various protein-SDS complexes carry the same density of negative charges. The negative charge of the protein-SDS complex far exceeds the original charge of the protein molecules, thus covering up the original charge differences between different types of proteins. At this time, the electrophoretic mobility of the protein molecule mainly depends on its relative molecular mass, while the influence of other factors on the electrophoretic mobility is almost negligible.

When the relative molecular mass of the protein is between 15,000 and 200,000, the electrophoretic mobility has a linear relationship with the logarithm of the relative molecular mass. If the mobility of a standard protein of known relative molecular mass is plotted against the logarithm of the relative molecular mass, a standard curve can be obtained. The unknown protein is electrophoresed under the same conditions, and the relative molecular mass can be calculated on the standard curve based on its electrophoretic mobility.

Plant Proteins

Procedures

1. Installation

Install a sandwich vertical plate electrophoresis tank.

2. Preparation of gel

Select the appropriate separation gel concentration according to the relative molecular mass range of the measured protein.

3. Preparation of Gel Plate

• Preparation of separation gel. Prepare 20 mL of 10% separating gel. After mixing, use a slender tip dropper to add the gel solution to the gap between the long and short glass plates, about 8 cm high. Use a 1 mL syringe to take a little distilled water and slowly inject 3-4 mm along the wall of the long glass plate to achieve water sealing. After about 30 min, a boundary with different refractive index appears between the gel and the water sealing layer, indicating that the gel is completely polymerized. Pour off the distilled water, and then use filter paper strips to absorb excess water.
• Preparation of stacking gel. Prepare 10 mL of 3% stacking gel. After mixing, use a slender tip dropper to add the stacking gel above the polymerized separation gel until it is about 0.5 cm away from the upper edge of the short glass plate. Gently insert the sample slot template into the stacking gel to avoid introducing air bubbles. The gel polymerizes after about 30 minutes and is left for another 20-30 min. After the gel solidifies, carefully remove the sample groove template, use narrow filter paper to absorb the excess water in the sample groove, and pour the pH 8.3 Tris-glycine buffer into the upper and lower storage tanks. It should cover the short plate by more than 0.5 cm, and you are ready to add samples.

4. Sample processing and loading.

Each standard protein and protein to be tested were dissolved in sample dissolving solution to a concentration of 0.5-1 mg/mL, heated in a boiling water bath for 3 min, and cooled to room temperature for later use. If the processed sample solution has been stored for a long time, it should be heated in a boiling water bath for 1 min before use to eliminate metastable polymerization.

The general sample volume is 10-15 µL (i.e. 2-10 µg protein). If the sample is thin, the adding volume can be increased. Use a microsyringe to carefully add the sample through the buffer to the bottom of the concave sample slots of the gel. When all concave sample slots are filled with samples, electrophoresis can begin.

5. Electrophoresis

Turn on the switch of the DC stabilized electrophoresis instrument and initially adjust the current to 10 mA. When the sample enters the separation gel, adjust the current to 20-30 mA. When the blue dye migrates to the bottom, turn the current back to zero and turn off the power. Unplug the fixed plate, take out the glass plate, use a blade to gently pry open a piece of glass and remove it, cut off a corner of one end of the gel plate as a mark, and move the gel plate to a large petri dish for staining.

6. Dyeing and decolorization

Pour the staining solution into the petri dish and stain for about 1 h. Rinse several times with distilled water, and then destain with destaining solution until the protein bands are clear, that is, use a ruler to measure the distance between each band and the top of the gel.

7. Calculation

Relative mobility (mR) = sample migration distance (cm)/dye migration distance (cm).

Plot the logarithm of the relative molecular mass of the standard protein against the relative mobility to obtain a standard curve. According to the relative mobility of the sample to be tested, its relative molecular mass is found from the standard curve.

Note

• Before installing the sandwich vertical plate electrophoresis tank, the rubber strips, glass plates, and tanks must be clean and dry; do not touch the glass on the gel-filled surface with your hands.
• Not all proteins can have their relative molecular mass measured by SDS-PAGE, and it has been found that the relative molecular mass of some proteins measured by this method is unreliable. Including: proteins with abnormal charge or conformation, proteins with larger prosthetic groups (such as certain glycoproteins), and some structural proteins. Therefore, it is best to use at least two methods to determine the relative molecular mass of unknown samples and verify each other.
• Many proteins are composed of subunits or two or more peptide chains, which are dissociated into subunits or single peptide chains under the action of SDS and mercaptoethanol. Therefore, for this type of protein, SDS-gel electrophoresis only measures the relative molecular mass of their subunits or single peptide chains, rather than the relative molecular mass of the complete molecule. In order to obtain more comprehensive information, other methods must be used to determine its relative molecular mass and the number of peptide chains in the molecule, etc., and cross-reference with the results of SDS-gel electrophoresis.

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