How to Measure the Isoelectric Point of Proteins

The isoelectric point (pI) of proteins is the pH value at which the protein is electrically neutral and has a net charge of zero in solution. Determining the pI of proteins is essential for understanding their chemical properties, purification, and analysis. Below are some commonly used methods to determine the pI of proteins.

 

Isoelectric Focusing (IEF)

Isoelectric focusing is a technique based on the principle that proteins do not migrate at their isoelectric point pH. In a gel with a pH gradient, the protein will focus at the location of its isoelectric point. The pI can be determined by knowing the protein's migration in the gel.

 

Two-Dimensional Gel Electrophoresis (2D-PAGE)

In 2D-PAGE, the first dimension is usually IEF, and the second dimension is SDS-PAGE. By comparing the protein's migration in two dimensions, information about the protein's molecular weight and isoelectric point can be obtained simultaneously.

 

Computational Prediction

Based on the protein's amino acid sequence, some online tools or software can be used to calculate the theoretical isoelectric point of the protein.

 

pH Differential Method

Taking advantage of the characteristic that proteins have a net charge of zero at their isoelectric point, observe changes in protein solubility or other properties in buffers of different pH. The pH point where the protein properties change significantly can be identified as the isoelectric point.

 

Capillary Isoelectric Focusing (cIEF)

cIEF is a capillary isoelectric focusing technique. In a pH gradient within a certain range, the protein will focus at its isoelectric point. This technique allows for accurate information about the protein's isoelectric point in a shorter time.