Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis

This method can help answering key questions regarding in tectonics of intrinsic disorder proteins such as conformational changes due to metal iron binding. The main advantage of this technique is that it requires very little equipment and the results can be obtained very quickly. Demonstrating the procedure will be Matthias Stein, a grad student from my laboratory.

To begin, use a glass cutter on a glass plate to cut a bare fused silica capillary with a polyamide external coating and an inner diameter of 50 microns into 33 and 30 centimeter lengths. Use a pen to mark the middle of a one centimeter wide detection window at a distance of 24.5 centimeters from one end of the capillary for the CGE experiment and at 21.5 centimeters from one end of the capillary for the ACE experiment. Using a blowtorch, burn off the polyamide external coating 0.5 centimeters before and after each mark.

Similarly, use the blowtorch to remove one centimeter of the coating at both ends of the capillaries. Then, use ethanol and a soft tissue to clean the ends of the capillaries and the detection windows. Install a capillary into the CE system with the detection window near the outlet.

After preparing solutions, according to the text protocol, use 250 micromolar calcium chloride solution to fill a 10 milliliter syringe. Then attach a 0.2 micron PVDF filter to the syringe and push 2 milliliters of the solution through the filter to discard it. Use the remaining calcium chloride in the syringe to fill 10 vials up to the maximum allowed volume.

Label each vial as 250 micromolar calcium chloride solution inlet vial. Next, using the calcium chloride solution, fill 10 vials halfway. Mark each as 250 micromolar calcium chloride solution outlet vial.

Then, with other metal salt containing solutions, fill vials in a similar manner. In addition, for each pair of inlet and outlet vials, use 30 micromolar Tris Buffer to prepare a similar set of inlet and outlet vials. With 60 micromolar acetanilide EOF marker, fill 10 vials.

Then use the one milligram per milliliter sample solution to fill one vial. After preparing the analysis, according to the text protocol, run the separation by hydrodynamically injecting the sample solution for the CGE experiments and applying 0.1 bar for four minutes at the inlet. Then, apply negative 16.5 kilovolts and a pressure of 2.0 bar at both ends of the capillary for 25 minutes.

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 asset 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. Alternately, repeat taking measurements with and without ligands. Then, repeat the method using the alkaline earth ligand solutions.

Finally, use the following solutions to carry out the same method. Then, calculate the change in charge size ratios for the various protein metal ion interactions. Shown here is the electropherogram for the AtHIRD11 sample obtained during the CGE experiments.

The peptide size increases from left to right. Peak number four has the largest mass and indicates the intact protein. The smaller peaks, two and three, represent smaller impurities.

This figure represents the electropherogram for acetanilide during the ACE experiments. The acetanilide solution shows only one high peak since it should have no impurities. The detection time for the peak maximum indicates the migration time of the electroosmotic flow and is used for the calculation of the interaction.

This electropherogram of the AtHIRD11 sample during the ACE experiment was performed in the absence of SDS and metal ions. Besides the two impurities from the CGE electropherogram, at least five peaks, which are related to the protein itself, are present. A graphical evaluation of the measured migration time shifts in the presence of the various metal ions for peak six, is shown here.

It is represented by the calculated value Delta R over RF.Each result indicates how strong of a shift has occurred by its value and its sign as indicated by the overall change in charge of the protein metal ion complexes. Once mastered, the technique can be done for one interaction partner in eight hours including the CGE interaction if it's performed properly. While attempting this procedure, it is important to remember to change the protein solutions for long time measurements since degradation products can increase over time and to change the Tris Buffer solutions in order to obtain more precise results.

After watching this video, you should have a good understanding of how to cut and clean a capillary, as well as, set up an affinity capillary electrophoresis method, in order to evaluate the binding behavior of intrinsic disordered proteins.