Protein Chemistry. Structure, Properties, and Research Methods - Shendryk A.N. 2022

Methods for Experimental Investigation of Protein Structure
Protein Separation Methods
Experimental Methods of Electrophoresis - Immunoelectrophoresis

This is a two-stage Electrophoresis method proposed in 1953 by Pierre Grabar. First, a mixture of substances is separated using gel electrophoresis, and subsequently, the Components of the mixture are detected via immunodiffusion. Immunoelectrophoresis can only be used to analyze components that undergo a precipitation reaction with Antibodies, which essentially means Proteins.

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Immunoelectrophoresis is performed on horizontal plates using Agar or agarose gels. A uniform layer of gel is applied to the plate, and two wells are formed within this layer: one in the shape of a well, and the other in the shape of a trough (see fig.). A mixture of antigen proteins is placed into the well, and standard electrophoresis is carried out. During electrophoresis, the proteins in the mixture migrate according to their molecular charges and distribute across the plate. Upon completion of electrophoresis, anantiserum containing antibodies is introduced into the trough. The antibodies from the serum and the Antigens distributed across the plate begin to diffuse toward one another. Upon meeting, the antigen and antibody form an insoluble antigen-antibody complex (precipitate), which settles out. This results in The formation of visible, curved ellipsoidal precipitation lines (arcs). THE POSITION OF these arcs on the plate is determined by the diffusion coefficient of the antigen, given that the antibodies have very similar mass and shape, and consequently nearly identical diffusion coefficients. The number of formed arcs corresponds to the number of distinct antigens present in the mixture. This method enables the detection and Separation of protein mixture components that share identical electrophoretic mobility but possess different diffusion coefficients.

An example of the Structure/182.html">Practical Application of EP for determining the pI of proteins

As is known, the pI of Amino Acids can be calculated if the pK_a values of their ionizable groups are known. However, the pI of a polypeptide cannot be determined in this manner. Therefore, to find the pI of Peptides, one can proceed as follows.

> Measure the mobility of the peptide at several different pH values.

> Plot a graph of mobility versus pH (see fig.).

> Determine from the graph the pH value at which the electrophoretic mobility equals zero.

Knowing the position of the isoelectric point for a protein is crucial when isolating it from a mixture, since the solubility of a protein is at its minimum at pH = pI.