Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes
Published: April 30, 2023
Abstract
Source: Nachbar M. et al., Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis. J. Vis. Exp. (2018).
This video describes the method to analyze protein-ligand interaction by mobility-shift affinity capillary electrophoresis. This method can be used for characterizing the binding behavior of a protein with various charged ligands.
Protocol
1. Prepare the solutions for the ACE analysis Prepare the 1 M NaOH solution. Weigh 4.0 g of NaOH in a 100 mL volumetric flask. Fill it up to the mark with deionized water to make a 1 M stock. Prepare the 0.1 M ethylenediaminetetraacetic acid (EDTA) in a 0.1 M NaOH solution. 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. Weigh 2.42 g of EDTA …
Disclosures
The authors have nothing to disclose.
Materials
| 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 |
Cite This Article
Mobility Shift Affinity Capillary Electrophoresis: A Method to Analyze Sample-Ligand Interactions Depending on Differential Migration of Protein-Ligand Complexes. J. Vis. Exp. (Pending Publication), e21118, doi: (2023).