Antibody Isoelectric Point Determination and Application in Biochemical Analysis

The isoelectric point (pI) of an antibody is the pH value at which the antibody does not migrate during electrophoresis. At this pH value, the positive and negative charges of the antibody are balanced, making it neutral overall. The determination of the isoelectric point is very important for understanding and analyzing proteins, especially antibodies, as it can affect the stability, solubility and biological activity of the antibodies.

 

Isoelectric focusing (IEF) is a commonly used technique for determining the isoelectric point of proteins. In isoelectric focusing, proteins are separated in a gradient pH gel according to their pI. This process is based on the net charge of the proteins at a specific pH. If the environmental pH is lower than the pI of the protein, the protein will be positively charged; if the environmental pH is higher than the pI of the protein, the protein will be negatively charged. During isoelectric focusing, under the action of an electric field, proteins migrate in the direction opposite to their charge until they reach a position where the pH equals their isoelectric point. At this position, the migration of the proteins stops because their net charge is zero.

 

The specific steps for determining the isoelectric point may vary depending on the equipment and reagents used, but generally include the following steps:

 

Sample Preparation

Extract the antibody or protein sample, which may need to be purified by various methods.

 

Gel Preparation

Use a special gel with a pH gradient, which is usually achieved by embedding a series of different pH buffers in a polyacrylamide gel.

 

Sample Application

Apply the protein sample to the gel.

 

Electrophoresis

Apply an electric field to separate the proteins in the gel according to their isoelectric points.

 

Detection and Analysis

Use specific staining or other detection methods to visualize the proteins, then analyze the results to determine the isoelectric point.