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

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

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.

Materials

List of materials used in this article

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