Protein Gel Electrophoresis Animation

1. D Projection Electrophoresis For Single Cell Immunoblotting
2. Electrophoresis: A Brief Primer
3. Two Dimensional Difference Gel Electrophoresis

Electrophoresis is a chemical process in which an electric charge in a solution flow toward an opposing electrode. In the 1930s, Swedish biophysicist Arne Tisselius developed electrophoresis while researching blood proteins. In 1948, Arne Tisselius received Novel Prize in Chemistry for his contributions to the electrophoretic technique.

Gel electrophoresis is one of the laboratory methods for separating DNA, RNA, or protein molecules based on their electric charge or size.

The principle behind electrophoresis is the observation that the majority of biomolecules exist as electrically charged particles with ionizable functional groups. A solution containing biomolecules will have either positively or negatively charged ions depending on the pH.

D Projection Electrophoresis For Single Cell Immunoblotting

When charged molecules are placed in an electric field, they travel in the opposite direction of the positive or negative pole. Depending on the mass and net charge of each particle in the solution, ionized biomolecules will migrate at different rates when exposed to an electric field. Negatively charged particles such as nucleic acids gravitate toward the anode, while the positively charged particles toward the cathode. Each charged particle will migrate in a pattern determined by its particular property due to changes in speed and direction, allowing for the separation of biomolecule components with similar properties.

Two types of buffers exist Acidic buffer and a Basic buffer. For a lower pH, acidic buffers, including citrate, acetate, formate, and phosphate, are utilized. Basic buffers like tric, borate, and tricine are employed to keep pH levels high. 

They are also categorized as native and denaturing, where RNA or proteins are kept in their native structure while running through the gel in native gel electrophoresis. In contrast, the RNA or protein are reduced to their linear structure before or during gel electrophoresis in denaturing gel electrophoresis. This reduction is achieved by the addition of a reducing agent to the sample, gel, and/ or buffer, which separates the bonds within the RNA or protein molecule and results in the formation of a secondary structure.

Electrophoresis: A Brief Primer

The pH level known as the isoelectric point is the one where proteins have no net charge (pI). Proteins are separated by their isoelectric points within a continuous pH gradient using the high-resolution approach known as isoelectric focusing (IEF). Compounds that differ in pI by only 0.01 pH units can be separated thanks to the excellent resolving power.

Gel solution preparation: A gel is prepared by dissolving it in boiling water. After cooling to a more comfortable temperature, the solution is poured into a mold or caster.

Gel casting: A comb is used to create wells in the gel once it has been set. The gel is then inserted into the electrophoretic chamber. Buffer fills the chamber to a maximum of one-third of its total volume.

Two Dimensional Difference Gel Electrophoresis

Sample preparation: To give the sample color and density, loading dye is added, which can be either a fluorescent tag or ethidium bromide.

The DNA is isolated and pre-processed, and placed in a solution with some basic blue dye to help visualize the movement of the sample through the gel.

Electrophoresis: The chamber and a power supply where the voltage is set are connected by the negative and positive leads, respectively. The electric field and negatively charged particles are created when the power supply is turned on. DNA that is negatively charged migrates toward the anode because molecules gravitate toward electrodes with opposing charges.

Gel Electrophoresis Principles And Applications

Stopping electrophoresis, Staining, and Visualization: Dye is used to following the migration visually. The power supply is turned off. The gel is stained and visualized using a gel imager when the procedure is finished. By comparing the size of the sample fragments to the standard, the logarithm of the molecular weight is used to calculate their sizes.

Prakriti Karki completed her B.Sc. in the field of Microbiology. She is interested in working in the interface of immunology, microbiology, synthetic biology, bioinformatics, and open science. She has worked as a project lead at Media Lab Nepal, as a research associate in the BMSIS program, and as an awareness community member at the iGEM WiSTEM initiative.

Sample preparation: To give the sample color and density, loading dye is added, which can be either a fluorescent tag or ethidium bromide.

The DNA is isolated and pre-processed, and placed in a solution with some basic blue dye to help visualize the movement of the sample through the gel.

Electrophoresis: The chamber and a power supply where the voltage is set are connected by the negative and positive leads, respectively. The electric field and negatively charged particles are created when the power supply is turned on. DNA that is negatively charged migrates toward the anode because molecules gravitate toward electrodes with opposing charges.

Gel Electrophoresis Principles And Applications

Stopping electrophoresis, Staining, and Visualization: Dye is used to following the migration visually. The power supply is turned off. The gel is stained and visualized using a gel imager when the procedure is finished. By comparing the size of the sample fragments to the standard, the logarithm of the molecular weight is used to calculate their sizes.

Prakriti Karki completed her B.Sc. in the field of Microbiology. She is interested in working in the interface of immunology, microbiology, synthetic biology, bioinformatics, and open science. She has worked as a project lead at Media Lab Nepal, as a research associate in the BMSIS program, and as an awareness community member at the iGEM WiSTEM initiative.