Isoelectric Focusing Electrophoresis to Determine Protein pI

Proteins are amphoteric electrolytes, their charge comes from the ionized groups of the amino acid side chain, which can dissociate in solutions of a certain pH value, thereby carrying some charge. When the protein solution is at a certain pH, the protein dissociates into positive and negative ions with equal tendencies, becoming ampholytic ions with a net charge of zero. This pH of the solution is called the isoelectric point (Isoelectric Point, pI) of the protein.

 

At the pI of proteins, due to the minimum electrostatic repulsion between molecules, the solubility is also minimal, and precipitation easily forms. At the same time, its viscosity, osmotic pressure, expansibility, and conductivity are all minimal. This property has important application value in protein separation, purification, and electrophoresis experiments. The determination of the pI of proteins is of great significance for understanding the properties of proteins, studying the interaction between proteins and other molecules, and optimizing the process of protein separation and purification.

 

Isoelectric focusing electrophoresis (Isoelectric Focusing, IEF) is a method of analysis based on the relationship between the migration rate of proteins in an electric field and its net charge. It is commonly used to determine the pI of proteins. In isoelectric focusing electrophoresis, the protein sample is loaded into a gel with a gradient pH value, and then separated by an electric field. At a specific pH value, the net charge of the protein is zero, and it stays at the corresponding position to form an isoelectric focusing band. By observing the position of the pI focusing band, the pI of the protein can be determined.

 

The Specific Operation Steps of IEF to Determine the pI of Proteins

1. Preparation

Choose a suitable isoelectric focusing gel, clean the electrophoresis tank, and prepare the protein sample.

 

2. Sample Preparation

Dissolve the protein sample in an appropriate buffer, and adjust the protein concentration to a suitable range.

 

3. Load Sample

Load the prepared protein sample into the loading hole of the isoelectric focusing gel.

 

4. Electrophoresis

Add an appropriate electrophoresis buffer to the electrophoresis tank, apply an electric field, and perform electrophoresis separation.

 

5. Staining and Observation

After electrophoresis, stain the gel and observe the location of the protein focusing band.

 

Because IEF has the advantages of high resolution, good repeatability, large sample capacity, and easy and fast operation, it has been widely used in biochemistry, molecular biology, and clinical medical research. MtoZ Biolabs has established a protein isoelectric point detection platform based on the IEF principle for various types of biological products. It can accurately measure the isoelectric point of proteins, antibodies, vaccines, peptides, collagen proteins, etc. We welcome your inquiries.