What is Protein Electrophoresis?

Instructor: Amanda Robb
This lesson is on protein electrophoresis, a common technique used in biochemistry and molecular biology. This article will discuss the definition, purpose, methods, and uses for protein gels.

Protein Electrophoresis - Definition

Protein electrophoresis is the process of separating samples of proteins based on size. With electrophoresis, proteins travel through a gel matrix, inside a small box, which is usually used in scientific labs. An electric current pushes the proteins through the gel. The current acts like a little helper in each lane, shoving the proteins to their equilibrium state, where they won't move anymore. The image below shows a protein gel box. The red clamps on the side hold the gel together. The clear gel inside is polyacrylamide, and it creates the matrix for the proteins to move through. The blue samples are the proteins. The top and bottom of the box contain leads to hook up to an electric current. The scientist is adding more samples using a small dropper called a pipette.

Loading a gel box with protein samples
Gel Box

Smaller proteins migrate faster and show up at the bottom of the gel. Larger proteins take a longer time, and show up at the top of the gel. Think of it like having a jar filled with marbles. If you added sand to the jar and shook it, the sand would move to the bottom of the jar quickly. If you added a larger particle, like rice, and shook the jar, the rice would take a longer time to fall to the bottom. Protein electrophoresis works the same way. Proteins move to the bottom of the jar based on how quickly they can make it through the maze of the gel.

Purpose

Protein electrophoresis is mainly used in labs to study the presence or absence of proteins in a cell. Sometimes, scientists want to know if changing conditions in cells or animals increases or decreases the presence of a certain protein. For example, scientists might add a chemical to cells that mimics a disease and then observe which proteins are made. This allows them to understand the molecular biology going on inside cells and possibly design treatments for different diseases. This process is analogous to a murder investigation. If the police have a suspect and want to know more about how he committed the crime, they might look for known associates to see who helped him. The suspect is like the disease or chemical and the associates are like the proteins being tested in electrophoresis. During the last step of protein electrophoresis, scientists can determine exactly which proteins are present in their gel.

Running the Gel

The first step for separating proteins in gel electrophoresis is to boil the proteins with a chemical called sodium didecasulfate (SDS). This breaks apart the bonds in the protein and coats the entire thing with a negative charge. This helps the protein move through the gel using the electric current. The sample is also given a dye, coomassie blue, to make it easier to observe the progression of proteins moving in the gel. They appear blue and stand out on the clear background. This helps scientists know when to stop the gel. Below is an image of sample with coomassie blue in it.

Coomassie blue solution caption=

Next, the protein samples are put into wells at the top of the gel. This is a place for the sample to sit until it starts to run through the gel. It helps to organize the proteins into lanes, like how runners are organized in a race. Next, the electric current is applied. The electric force pushes the protein sample through the gel. After the proteins are sufficiently separated, the scientists will figure out which proteins are present. Below is a diagram of how to load a gel and run it. In the last step, notice how there are bands of proteins at the bottom. These ones are smaller, so they managed to get through the gel faster.

Steps to running a protein electrophoresis gel
protein electrophoresis steps

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