Separation of Proteins by Two-dimensional Gel Electrophoresis

Two-dimensional electrophoresis is a protein detection technology with higher resolution that combines isoelectric focusing electrophoresis and SDS-PAGE. The gel after two-dimensional electrophoresis is stained and presents a two-dimensional distribution diagram. The horizontal direction reflects the difference in the isoelectric point of the proteins, while the vertical direction reflects the difference in their relative molecular mass.

Principle

The first dimension of two-dimensional electrophoresis of proteins is isoelectric focusing (IEF), which separates proteins based on their different isoelectric points. The second dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates protein subunits according to their relative molecular mass. After two separations of charge and relative molecular mass, the isoelectric point and relative molecular mass information of the protein molecule can be obtained.

Isoelectric focusing (IEF) is a special type of polyacrylamide gel electrophoresis, which is characterized by adding an ampholyte carrier to the gel, so that the gel forms a continuous pH gradient in the electric field. Protein is a typical amphoteric electrolyte molecule. It moves toward the positive electrode of the electric field in the form of anions in a pH environment greater than its isoelectric point, and moves toward the negative electrode in the form of cations in a pH environment smaller than its isoelectric point. This swimming movement only stops in a pH environment equal to the isoelectric point. If a mixed sample of proteins containing various isoelectric points is electrophoresed in a pH gradient environment, then under the action of the electric field, regardless of the original distribution of this mixed group of protein molecules, each protein molecule will aggregate at the position corresponding to the pH gradient according to the size of their respective isoelectric points. After a certain period of time, different protein components are divided into different regions. This process is called isoelectric focusing. The charge of the protein at the site where the protein aggregates is zero. By measuring the pH of this site, the isoelectric point of the protein can be known.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is mainly used to determine the relative molecular weight of protein subunits. SDS is an anionic detergent. As a denaturant and cosolvent, it can break intramolecular and intermolecular hydrogen bonds, unfold molecules, and destroy the secondary and tertiary structures of protein molecules. Strong reducing agents (DTT, dithiothreitol) can cleave the disulfide bonds between cysteine residues. After adding SDS and reducing agents to the sample and gel, the molecules are depolymerized into their polypeptide chains. The depolymerized amino acid side chains are fully combined with SDS to form negatively charged protein-SDS micelles. The negative charge greatly exceeds the original charge of the protein molecules, which eliminates the original charge difference between different molecules. Therefore, the electrophoretic mobility of this micelle in the SDS-polyacrylamide gel system is no longer affected by the original charge of the protein, but mainly depends on the relative molecular mass of the protein or subunit. When the relative molecular mass of the protein is between 15-200 kD, the electrophoretic mobility has a linear relationship with the logarithm of the relative molecular mass.

Protein two-dimensional electrophoresis is a technology that combines the two characteristics of protein isoelectric point and relative molecular mass for protein separation. Therefore, it has high resolution and sensitivity, and has become a powerful biochemical method for the detection and analysis of proteins, especially proteins in complex systems.

Plant Proteins

Procedures

1. First-dimensional isoelectric focusing electrophoresis

a. Take out the frozen sample, add corresponding amounts of DTT, IPG Buffer and hydration solution to a final volume of 340 µL (18 cm IPG strip), vortex and mix thoroughly, and then centrifuge at 20°C and 20,000 g for 15 min.

b. Take the supernatant and spread it flatly in the strip tank, then slowly peel off the protective film of the IPG strip from the acid end (tip) side of the strip. Put the rubber side down and the positive end toward the tip of the strip slot into the strip slot. Slowly press down on the strip and move it up and down to avoid generating bubbles. Finally, put down the flat end of the IPG strip to allow the hydration solution to soak the entire strip, and ensure that both ends of the strip are in contact with the electrodes at both ends of the groove.

c. Cover the IPG strip with an appropriate amount of IPG Cover Fluid, completely cover the strip and liquid, and close the lid.

d. The tip back electrode of the strip groove is in contact with the anode platform of the IPGphor instrument, and the flat end is in contact with the cathode.

e. Set the operating parameters of the IPGphor instrument. The strip is first hydrated at 30 V and 20°C for 12 h and then enters the isoelectric focusing stage. Set the operating parameters of the instrument.

2. Balance of IPG strips

a. DTT balance: Take out the focused strip and put it into a balance tube, place the supporting surface against the inner wall of the test tube, add a balance buffer containing 1% DTT and shake slowly on a shaker for 15 min.

b. IAA (iodoacetamide) balance: Take out the first balanced strip and gently dip it in the electrophoresis buffer for a few times, then put it into a balance solution containing 2.5% IAA for 15 min (be careful to avoid light).

3. Fill two-dimensional gel

a. Select an appropriate vertical electrophoresis tank for the second dimension based on the size of the IPG strip during IEF electrophoresis in the first dimension. Generally, experiments use 10% or 12.5% separation gel and 5% concentration gel. Install the vertical electrophoresis system according to the instructions, and prepare the separation gel in a 500 mL beaker.

b. Fill the separating gel and stacking gel separately.

c. The poured gel is capped with water-saturated n-butanol to make the gel surface as smooth as possible.

d. After the gel has solidified, pour away the water-saturated n-butanol on the top, gently wash the gel surface twice with deionized water, and then wash it with electrophoresis buffer.

4. Transfer and sealing

The balanced gel strip was taken out of the test tube and washed three times with electrophoresis buffer. Add the pre-thawed 0.5% agarose capping solution to the glue surface, and quickly push the glue strip into it so that the lower edge of the glue strip is in close contact with the glue surface. There should be no air bubbles between the rubber strip and the rubber surface. After the agarose capping solution solidifies, place the glass plate into the electrophoresis tank.

5. Second dimension: SDS-PAGE

a. Turn on the condensed water circulation device 45 minutes before performing second-dimensional electrophoresis so that it reaches the preset temperature (10°C) during electrophoresis. Install the glass plate onto the vertical electrophoresis apparatus, add 1X electrophoresis buffer, and start electrophoresis.

b. Electrophoresis until the bromophenol blue reaches the bottom of the gel, then turn off the power, carefully remove the gel, mark it, place it in a staining tray and wash with deionized water for 5 min.

6. Staining

a. Silver staining

Fixation: Put the gel into the stationary liquid overnight, then wash it twice with deionized water, 10 min each time.

Sensitization: Place the glue in the sensitizing solution for 30 min, and then wash it with deionized water 3 times, 5 min each time.

Silver staining: Dye in silver staining solution for 20 min (protect from light), then wash with MilliQ water twice, 1 min each time.

Color development: Place the gel in the color development solution and shake gently for about 5 min.

Stop development: Place the gel in the stop development solution for 10 min, and then wash it twice with deionized water, 5 min each time.

b. Coomassie brilliant blue staining: Wash the peeled gel with deionized water for 10 min, then place it in the staining solution and stain it overnight on a decolorizing shaker. Then rinse with rinse solution 3-4 times, about 1 h each time, until the background color is lighter.

7. Image scanning and analysis

Carefully place the glue on the glass plate of the image scanning system, with one side parallel to the edge of the glass plate, taking care to avoid bubbles and water marks. The scanner should be set to the appropriate resolution and contrast.

Note

• Sample pretreatment is the key to two-dimensional electrophoresis. Samples for isoelectric focusing electrophoresis must remove salt ions, pigments, phenols, nucleic acids and other substances, and inhibitors to prevent proteolysis must be added. Therefore, relevant information must be consulted for sample pretreatment according to different samples.
• Determine the concentration of the gel used based on the relative molecular mass range of the pre-separated protein.
• There are many gel staining methods. The main difference is the sensitivity, which needs to be selected according to actual needs. Usually Coomassie brilliant blue staining can detect protein spots with a content of approximately 1 µg, and silver nitrate staining can detect protein spots with a content of ng.
• Repeatability is one of the issues that need to be paid attention to in two-dimensional electrophoresis. Therefore, the consistency of reagents, operating procedures, and experimental conditions should be maintained during the operation to ensure the repeatability of two-dimensional electrophoresis and thereby obtain reliable comparability.

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