Experiments for the determination of the isoelectric point of proteins
Experiments for the determination of the isoelectric point of proteins
The isoelectric point of a protein is one of the important properties of proteins. Determination of isoelectric point of proteins is important for understanding the physicochemical properties of target proteins and designing strategies for purification of target proteins. Currently, there is one main method used to determine the isoelectric point of proteins: isoelectric focusing electrophoresis to determine the isoelectric point of proteins.
Principle
Determination of protein isoelectric point by isoelectric focusing electrophoresis The basic principle of isoelectric focusing electrophoresis (IFE) is to use a polyacrylamide, agarose or dextran gel as a support and add a carrier amphoteric electrolyte (CE) to the gel.
The carrier amphoteric electrolyte is a mixture of aliphatic polyamino and polycarboxylic groups, which can naturally form a continuous linear pH gradient in the electric field with the positive pole being acidic and the negative pole being alkaline .
When protein molecules are electrophoresed in this continuous linear pH gradient, they move to the positive pole as anions in pH environments larger than their isoelectric point, and move to the negative pole as cations in pH environments smaller than their isoelectric point, and finally accumulate at the corresponding position of their isoelectric point, from which the isoelectric point of a protein can be determined.
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