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

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.

After preparing the method for the measurements without ligands, prepare the method for the measurements with ligands, by first using 0.1 molar EDTA solution to rinse the capillary at 2.5 bar for 1 minute. Then, use deionized water to rinse the capillary. Next, equilibrate the capillary by using ligand solution to rinse it at 2.5 bar for 1.5 minutes.

Then, inject the acetanilide solution at 0.05 bar for 6 seconds, and change the inlet and outlet vials to the ligand-containing buffer vials. Apply 0.05 bar for 2.4 seconds, in order to push the acetanilide solution further in from the tip of the capillary. Apply 10.0 kilovolts for 6 minutes, and detect the acetanilide peak at a wavelength of 200 nanometers.

After rinsing the capillary with EDTA, deionized water, and the ligand solution as before, inject the protein sample, and change the inlet and outlet vials to fresh ligand-containing buffer vials. Ultimately, repeat taking measurements with and without ligands.