Protein Electrophoresis is a technique that is primarily utilized as a part of identifying proteins and separating them based on the point of charge that they have. It can be said that the electrical variance that exists in a protein will be dependent on several factors like the number of ionizable groups in a protein, the pH of its solution, and type of dye employed for staining. The technique was originally used to categorizes bacteria but its use has become extensively widespread ever since.
What Color Tube For Protein Electrophoresis?
The color of the gel during protein electrophoresis is determined by the buffer used to prepare the gel. If you have a high performance liquid chromatography (HPLC) machine, the buffers are specifically designed for that purpose.
But if you don’t have a HPLC machine, you can use the standard buffers available from several manufacturers and adjust them to get the color of your choice. Since you are using proteins, you want to match the color of your gel with that of your protein.
What is Protein Electrophoresis?
Protein electrophoresis is a laboratory technique for separating complex mixtures of macromolecules, such as proteins or DNA fragments, by size and charge. A gel is placed between the two electrodes of an electrical source. This gel has been previously soaked in a solution containing the molecules to be separated, called the sample buffer.
The protein molecules are then caused to migrate through the gel towards the positive electrode (anode) by applying an electrical current. Depending on their size and charge, the different molecules travel through the pores of the gel at different speeds.
Because most proteins have a negative charge on their surface, they are attracted to the positive electrode (anode). DNA fragments, which tend to be smaller than proteins, are not charged. As a result, they do not migrate with an applied electric field, but remain trapped in the pores of the gel.
How does Protein Electrophoresis work?
The procedure involves placing the protein solution under a non-polarizing electric field. The negative charges of the proteins will cause them to move towards the positively charged anode, while the positive charges from the proteins will cause them to be repelled by the negatively charged cathode.
In this case, the anode is at the top and cathode at the bottom. The proteins are separated into bands based on their charge and size that migrate throughout the gel at different speeds.
The amount of time it takes for each band to reach its point of equilibrium as well as where they are located in relation to each other can then be used to identify any abnormalities in their structure, shape or size since it may have been altered due to genetic mutation or recombination.
What is the significance of Protein Electrophoresis?
Protein electrophoresis is a technique used to separate proteins based on their electric charge and size. When a protein is placed in an electric field, it will move toward the positive or negative end of the field. The rate at which the protein moves depends on its mass and electrical charge. This movement can be measured and used to determine the approximate size of a protein and how much it is charged.
Proteins are large molecules made up of one or more chains of amino acids. These amino acids come in two varieties: positively charged, or basic, and negatively charged, or acidic. At any given time, some of the protein’s chains will have a net positive charge while others will have a net negative charge. The rate at which these chains move depends on their number of basic amino acids and total mass. So proteins that have similar structures but different masses will travel at different speeds in an electric field because the heavy ones will be more resistant to the electric field than lighter ones.
The mass of a protein is typically determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which uses an electrical current to determine how long it takes for a protein to move through an electric field.
Phases of Electrophoresis
There are two major steps in this process:
1. SDS-PAGE
The sample must be mixed with a detergent called sodium dodecyl sulfate (SDS), which breaks down the protein structure by unfolding each protein strand and exposing its hydrophobic parts. The detergent then binds with the free hydroxyl groups on each protein, reducing its net charge and preventing it from binding with other non-detergent molecules in the sample, such as DNA. This is called denaturing the protein.
2. Electrophoresis
The sample is placed into an electrical field, which separates proteins based on size and shape. The smaller proteins move faster than larger proteins through the electric field, so they reach their point of equilibrium first, at which point movement cannot continue until there is another change in charge or electrical field.
What are the components of electrophoretic method?
There are three basic components of the electrophoresis:
- The gel. A gel is a solid substance that is used to separate the proteins. The gel is made up of a protein known as agarose, which is obtained from seaweed. When the agarose is mixed with water, it forms a viscous solution that can be poured into a container and allowed to set. The proteins are then placed in this gel solution to separate them according to their size and charge.
- The buffer. This component of electrophoresis is a weak acid or base solution used to regulate the pH level in the gel. It also helps to maintain a constant electric field during electrophoresis. The buffer may have a pH level between 3-10 or higher depending on the type of protein being analyzed and how many charges it carries on its surface. Proteins that have an acidic charge will move more slowly through the buffer when compared to proteins that have a basic charge since they respond differently to electric current flow through their environment.
- The electrodes (stainless steel rods). These rods are connected at either end by wires that carry an electrical current through them when they are submerged in water or another liquid solution like buffer or DNA stain solution which contains
Last Words
Protein electrophoresis is a normal process that can be used to determine protein levels in your body. This test is performed in order to determine if there are any abnormal proteins present in your blood. These abnormal proteins can be caused by a number of factors including infection, inflammation or other diseases.
Protein electrophoresis works by separating the proteins of your blood into different groups based on their size and charge. Each group will have a different mobility, which means that some proteins will move faster than others during the process. The speed at which each protein moves depends on its size and charge, as well as the amount of salt present in the sample.