Encyclopedia of Experiments
Biological Techniques
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Encyclopedia of Experiments Biological Techniques
Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes

Transkript

Ligands, such as metal ions, bind non-covalently to a specific protein, forming a protein-metal ion complex. This binding alters the overall charge of the protein, which allows for the characterization of these complexes using affinity capillary electrophoresis.

To begin, take a thin, pre-conditioned glass capillary with charged silanol groups on the inner surface. Rinse the capillary with EDTA solution. EDTA is a chelating agent that removes any metal ion impurities. Now, hydrodynamically inject the sample solution containing the desired protein into the capillary from its positive end or anode. 

Apply a high voltage to create an electro-osmotic flow, forcing the proteins in their native conformation with inherent charges to move inside the capillary toward the cathode. Record the migration pattern of unbound proteins from the capillary.

Next, introduce a specific ligand solution into the capillary along with the protein samples, and apply the same voltage. Inside the capillary, the ligand molecules bind non-covalently to the target protein, forming complexes.

This induces a conformational change in the proteins, leading to an alteration in their inherent charges and, impacting the charge-to-size ratio. These changes modulate their interactions with the charged surface of the capillary, causing them to flow differently compared to the unbound protein.

Record these changes in the electrophoretic mobility, which correlate with the strength of the protein-ligand interaction.

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