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

プロトコル

1. Prepare the solutions for the ACE analysis

1. Prepare the 1 M NaOH solution.
   1. Weigh 4.0 g of NaOH in a 100 mL volumetric flask.
   2. Fill it up to the mark with deionized water to make a 1 M stock.
2. Prepare the 0.1 M ethylenediaminetetraacetic acid (EDTA) in a 0.1 M NaOH solution.
   1. Transfer 10 mL of 0.1 M NaOH in a 100 mL volumetric flask using a 10 mL bulb pipette. Fill it up to the mark with deionized water.
   2. Weigh 2.42 g of EDTA on a weighing boat and dissolve the solid compound in the 100 mL of 0.1 M NaOH.
3. Prepare the Tris buffer (30 mM) solution.
   1. Add 3.63 g of Tris, 200 mL of deionized water, and a stir bar in a 500 mL beaker. Place the beaker on a stir plate and switch it on.
   2. Put a pH meter electrode in the beaker and adjust the pH to 7.4 using 0.1 M HCl.
   3. Fill the solution in a 1 L volumetric flask and fill up to the mark with deionized water.
      NOTE: This procedure has to be repeated or a bigger volumetric flask is needed since more than 1 L is needed for the whole analysis.
4. Prepare the acetanilide electroosmotic flow (EOF)-marker (60 µM) solution.
   1. Add 6 mg of acetanilide and 100 mL of Tris buffer in a 100 mL volumetric flask.
   2. Put the volumetric flask in an ultrasonic bath and switch it on for 30 min.
5. Prepare the sample (1 mg/mL) solution.
   1. Put a 0.5 mL microcentrifuge tube on a precision balance and add 0.3 mg of freeze-dried AtHIRD11 powder into the tube.
   2. Use a micropipette to add 0.3 mL of a 30 mM Tris buffer (pH 7.4) in the tube. Shake the tube carefully to dissolve the protein.
6. Prepare the ligand solutions.
   1. Prepare the CaCl₂ (5 mM) stock solution.
      1. Take a weighing boat, put it on an analytical balance and weigh 36.76 mg of CaCl₂*H₂O.
      2. Using a micropipette, flush the CaCl₂*H₂O from the weighing boat into a 50 mL volumetric flask with the previously prepared Tris buffer.
      3. Fill the volumetric flask with the Tris buffer up to the mark.
   2. Prepare the remaining stock solutions (5 mM).
      1. Repeat step 1.6.1 using the other metal salts instead of CaCl₂*H₂O [MgCl₂: 23.80 mg, BaCl₂: 26.02 mg, Sr(NO₃)₂: 52.91 mg, MnCl₂: 31.46 mg, SeCl₄: 52.91 mg, CoCl₂*2H₂O: 41.47 mg, CuCl₂*2H₂O: 42.62 mg, ZnCl₂: 3.41 mg, and NiCl₂*6H₂O: 59.43 mg].
         NOTE: The ZnCl₂ solution has a concentration of 0.5 mM. Since strong interactions between the ions and the inner capillary wall were observed, the concentration was reduced by a factor of 10⁴.
   3. Prepare the 500 µM CaCl₂ solution.
      1. Fill 10 mL of the CaCl₂ stock solution and put it in a 100 mL volumetric flask.
      2. Fill the volumetric flask with a Tris buffer to the mark and shake the flask.
   4. Prepare the 250 µM CaCl₂ solution.
      1. Fill 5 mL of the CaCl₂ stock solution and put it in a 100 mL volumetric flask.
      2. Fill the volumetric flask with a Tris buffer to the mark and shake the flask.
   5. Repeat previous steps for the other stock solutions.
7. Fill the solutions in the vials.
   NOTE: Every repeated run needs a set of inlet and outlet vials. The use of fresh ligand solutions at the inlet and outlet reduces the shifts in migration time after each run.
   1. Fill the 250 µM CaCl₂ solution in a 10 mL syringe.
   2. Put a 0.22 µm PVDF filter on the syringe and push 2 mL of the solution through the filter using the syringe in order to discard this filtered solution.
   3. Fill 10 vials up to the maximum allowed volume with the remaining 250 µM CaCl₂ solution of the syringe. Mark each as 250 µM CaCl₂ solution inlet vial.
   4. Fill 10 vials until they are half-filled with the 250 µM CaCl₂ solution. Mark each as 250 µM CaCl₂ solution outlet vial.
   5. Repeat steps 1.7.1 - 1.7.4 for the other metal salt-containing solutions.
   6. Repeat the steps for every pair of inlet and outlet vials using the 30 mM Tris buffer (pH 7.4) solution instead.
      NOTE: The 30 mmol/L Tris buffer pH 7.4 is used in between the runs to obtain migration time data for the protein in absence of metal ions. It is necessary to neglect changes in the EOF.
   7. Repeat the above steps for one vial using the acetanilide EOF-marker (60 µM) solution instead. Also, repeat these steps using the sample (1 mg/mL) solution instead.

2. Affinity Capillary Electrophoretic Analysis

1. Condition the capillary
   1. Set the thermostat to 23 °C.
      NOTE: The temperature is used according to published protocols and can be changed to other temperatures. However, the interactions are dependent on the temperature and will change accordingly.
   2. Flush a capillary for 40 min at 2.5 bar with 0.1 M NaOH.
   3. Flush the capillary for 10 min at 1.0 bar with deionized water.
   4. Flush the capillary for 30 min at 1.0 bar with a 30 mM Tris buffer (pH 7.4).
2. Preparation for the ACE methods
   1. Prepare the method for the measurements without ligands.
      NOTE: Acetanilide was not added to the sample solution, as it disguises 2 protein peaks.
      1. Rinse the capillary for 1 min at 2.5 bar with a 0.1 M EDTA solution.
      2. Rinse the capillary for 1 min at 2.5 bar with deionized water.
      3. Rinse the capillary for 1.5 min at 2.5 bar with a Tris buffer for equilibration.
      4. Inject the acetanilide solution for 6 s at 0.05 bar and change the inlet and outlet vials to the Tris buffer vials.
      5. Apply 0.05 bar for 2.4 s in order to push the acetanilide solution from the tip of the capillary further inside.
      6. Apply 10.0 kV for 6 min and detect the acetanilide peak at a wavelength of 200 nm.
      7. Repeat steps 2.2.1.1. - 2.2.1.6. using the protein sample instead of the acetanilide solution and detect all the protein peaks.
   2. Prepare the method for the measurements with ligands.
      1. Rinse the capillary for 1 min at 2.5 bar with a 0.1 M EDTA solution.
      2. Rinse the capillary for 1 min at 2.5 bar with deionized water.
      3. Rinse the capillary for 1.5 min at 2.5 bar with the ligand solution for equilibration.
      4. Inject the acetanilide solution for 6 s at 0.05 bar and change the inlet and outlet vials to the ligand-containing buffer vials.
      5. Apply 0.05 bar for 2.4 s in order to push the acetanilide solution from the tip of the capillary further inside.
      6. Apply 10.0 kV for 6 min and detect the acetanilide peak at a wavelength of 200 nm.
      7. Repeat steps 2.2.2.1 - 2.2.2.6 using the protein sample instead of the acetanilide solution and detect all the protein peaks.
   3. Alternately repeat steps 2.2.1 and 2.2.2 in order to calculate the change in the charge-size ratios for the various protein-metal ion interactions.
      1. Change the protein solution after every 60 h to a fresh one.
         NOTE: At this point, the experiment can be paused. This procedure is at least repeated 10x for each concentration of the ligands since the peak pattern varies slightly from run to run. If the solution is used longer than 60 h, the variations become too large.

資料

Name	Company	Catalog Number	Comments
AtHIRD11 sample	Shizuoka University (Group Prof. M. Hara)		Dehydrin Protein from Arabidopsis thaliana expressed in Escherichia coli
Barefused silica capillary,	Polymicro Technologies (Phoenix, USA)	106815-0017	TSP050375 50 μm inner diameter, 363 μm outer diameter, polyimide coating
Agilent 1600A	Agilent Technologies (Waldbronn, Germany)		Capillary electrophoresis instrument; Agilent 7100 CE can be used instead
Bulb pipette 25 mL	Duran Group GmbH(Mainz, Germany)	24 338 14	Preparing the ligand solution
Duran glas volumetric flask 25 mL	Duran Group GmbH(Mainz, Germany)	24 671 1457	Preparing the ligand stock solution
Duran glas volumetric flask 10 mL	Duran Group GmbH(Mainz, Germany)	24 671 1054	Preparing the ligand stock solution
Acetanilide	Sigma-Aldrich (Steinheim, Germany)	397229-5G	Electroosmotic flow marker
Manganese(II) chloride	Sigma-Aldrich (Steinheim, Germany)	13217	Ligand
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich (Steinheim, Germany)	431788-100G	Rinsing ingredient
Bariumchloride	Sigma-Aldrich (Steinheim, Germany)	202738-5G	Ligand
Nickel(II) chloride hexahydrate	Sigma-Aldrich (Steinheim, Germany)	654507-5G	Ligand
Selenium(IV) chloride	Sigma-Aldrich (Steinheim, Germany)	323527-10G	Ligand
2-amino-2-hydroxymethylpropane-1.3-diol (Tris)	Sigma-Aldrich (Steinheim, Germany)	252859-100G	Buffer ingredient
Zinc(II) chloride	Merck Millipore ( Darmstadt, Germany)	1088160250	Ligand
Strontium nitrate	Merck Millipore ( Darmstadt, Germany)	1078720250	Ligand
Calcium chloride dihydrate	Merck Millipore ( Darmstadt, Germany)	1371015000	Ligand
Copper(II) chloride dihydrate	Riedel-de Haën (Seelze, Germany)	31286	Ligand
Agilent ChemStation Rev. 8.04.03- SP1	Agilent Technologies (Waldbronn, Germany)	G2070-91126	Software packages to operate the CE instruments, acquisite data and evaluate it