Q&A of Two-Dimensional Gel Electrophoresis (2-DE)

A: The detection sensitivity of modern mass spectrometers and chromatographs is very high. As long as the protein spots are clearly visible to the naked eye (including the silver-stained spots), they are sufficient for protein identification. However, it is necessary to take care of a larger gel range and to take as many clear protein spots as possible to increase the success rate of mass spectrometry.

A: Commonly used reagents in proteolytic solution include urea, thiourea, CHAPS, DTT, IPG buffer, etc. Urea and thiourea are releasing agents (denaturing agents).

High concentrations of urea and thiourea can break the hydrogen bonds formed between protein molecules and prevent protein aggregation caused by hydrogen bonds and the formation of secondary structures during protein migration.

CHAPS is an amphoteric surfactant (detergent) that dissolves hydrophobic groups and enhances the solubility of proteins at their pI values.

The main component of IPG buffer is a carrier amphiphilic electrolyte with ionic properties, which keeps certain proteins that need to be soluble in the presence of salt ions more soluble.

A: Generally, when pressurized active hydration, the added voltage is very low (about 50V), and the resistance of the gel adhesive strip is relatively large, resulting in a current of less than 1uA, which is lower than the minimum display value of the instrument, so the current is displayed as zero (actually there is a trace of current). When the subsequent voltage is gradually increased, the current will also gradually increase and be displayed. In addition, if the current is zero at the beginning, from another perspective, it also indicates that the ion content in the sample is relatively low, and it is generally possible to carry out the subsequent iso-point focusing process smoothly.

A: The voltage of isoelectric focusing cannot rise to the specified value mainly because the ionic content of the sample is too high, resulting in a strong conductivity of the gel. To solve the problem, the ion content of the sample can be reduced by appropriate modification of the extraction method and the use of strategies such as membrane filtration and acetone precipitation. Excess salt ions can also be removed by using salt bridging in the step before isoelectric focusing as well as slow pressure boosting.

A: There are many reasons for the appearance of vertical streaks, and the phenomenon caused can vary. For example, horizontal streaks caused by proteins that are not adequately coated by SDS can occur throughout the entire area, and can be solved by extending the equilibration time and configuring the solutions accurately; incorrect pH values for Tris-HCL can also cause vertical streaks, which appear as rain and have a clear demarcation line in the upper center, and can be solved by configuring the pH accurately. In addition, the presence of impurities in the sample, such as DNA, can also cause streaking.

A: Both incomplete focusing and overfocusing will produce horizontal stripes. Incomplete focusing results in thicker and blurrier horizontal stripes, while overfocusing results in finer and clearer horizontal stripes.

Impurities in the sample can also cause cross-streaks.

A: Generally, it is possible. However, when the sample volume is very large, some proteins may not be absorbed by the glue strip, so there will be a lot of horizontal streaks after running 1D and 2D glue. In this case, it is better to transfer the glue strip to another electrophoresis tank for electrophoresis after re-soaking and swelling.

A: This is because BioRad's electrophoresis bath has a lid. In order to hold the gel strip in the electrophoresis tank, this lid has a corresponding protrusion designed to hold down the gel strip. Due to siphoning, this protrusion directs the mineral oil to the adjacent empty electrophoresis tank, thereby lowering the mineral oil level in the tank with the gel strip. If the gel strip is exposed to air, the effect on isoelectric focusing will be devastating. To prevent this from happening, an appropriate amount (80% full) of mineral oil can be added to the adjacent empty electrophoresis tank as well.

A: The square of the current is proportional to the power. As the current increases, the power increases and the heat released increases, which leads to an increase in the temperature of the glue strip. When the temperature exceeds 30 degrees Celsius, the urea in the buffer will tend to dissociate and produce some polar molecules, which will have an effect on isoelectric focusing.

A: At the beginning, there are more charged small molecules in the system (such as inorganic salts and bipolar molecules). During this phase, the current is mainly generated by the movement of these small molecules. These molecules have small masses and do not require high voltages to move them. When these small molecules move to their destination (inorganic salts to the opposite polarity electrode, amphiphiles to the corresponding pH strip), the proteins in the system start to take on the task of carrying the current and gradually move to the corresponding pH region.

A: The blue strip is produced by traces of bromophenol blue in the buffer being focused. Bromophenol blue is also a pH indicator. When it moves to the acidic region (pH 4), the color changes to yellow. The movement of bromophenol blue occurs roughly after the focusing of the polar small molecules and before the focusing of the protein macromolecules.

A: When the sample volume is relatively large or when the system has more salts, it is possible that the focusing voltage does not reach the set value.

A: When the upper sample volume is relatively small, all proteins move to their corresponding pH area values in a relatively short period of time, thus becoming neutral molecules. The resistance of the system is getting higher and higher, and the current becomes smaller and smaller at a constant voltage.

A: When the electro-focusing is finished, all the proteins move to the corresponding pI value region and become the central molecule. The voltage applied to the system then starts to electrolyze the water molecules, producing oxygen at the anode and hydrogen at the cathode.

A: The role of thiourea is to increase the solubility of proteins, especially basic proteins. The role of bipolar molecules is also to increase the solubility of proteins. When the protein moves to the corresponding pH value, it becomes a neutral molecule. The uncharged protein molecules tend to aggregate, thus reducing their migration efficiency during subsequent diastereotropic gelling, which may result in vertical off-tailing. Thiourea and bipolar small molecules will identify the interactions between neutral proteins and prevent their aggregation.

A: Calibration can be done with 2D gel standard proteins from BioRad, or with known proteins in the system for comparison.

A: The horizontal off-tailing may be due to 1) incomplete isoelectric focusing, 2) certain proteins themselves (glycoproteins), and 3) too high protein abundance.

The vertical tailing may be due to poor solubility of the protein when running two-way.

A: Nucleic acid, salt, detergent, etc.

A: The sample volume depends on the sample. If there are many proteins in the sample, the sample size should be larger so that there is enough for each spot to be detected. For a typical whole cell lysis system, the sample size should be between 100 µg (silver stain) and 500 µg (koil stain).