Name: analysis of athird11 intrinsic disorder and binding towards metal ions by capillary gel electrophoresis and affinity capillary electrophoresis
Uploaded: 2018-08-22T14:30:00
Description: Watch this Scientific Journal Video about Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis at JoVE.com

We grateful thank Masakuza Hara (the Research Institute of Green Science and Technology, University of Shizuoka, Japan) for providing the AtHIRD11 protein samples.

1. Preparation of the Capillary Electrophoresis Instruments
<ol>
	<li>Prepare the capillaries
	<ol>
		<li>Use a glass cutter to cut a bare-fused silica capillary with a polyimide external coating and an inner diameter of 50 &#181;m into 33 cm (for the CGE experiment) and 30 cm (for the ACE experiment) long capillaries. Perform the cutting on a glass plate. 
		Note: The 2 different experimental setups were developed for 2 different instruments. In order to use them on other instruments, a proper me...

<ol>
	<li>Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctionality+of+dehydrins:+An+overview.">The multifunctionality of dehydrins: An overview.</a> Plant Signaling &amp; Behavior. 5 (5), 1-6 (2010).</li><li>Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dancing+protein+clouds:+the+strange+biology+and+chaotic+physics+of+intrinsically+disordered+proteins.">Dancing protein clouds: the strange biology and chaotic physics of intrinsically disordered proteins.</a> Journal of Biological Chemistry. 291 (13), 6681-6688 (2016).</li><li>Hara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+the+Arabidopsis+KS-type+dehydrin+protein,+whose+gene+expression+is+constitutively+abundant+rather+than+stress+dependent.">Biochemical characterization of the Arabidopsis KS-type dehydrin protein, whose gene expression is constitutively abundant rather than stress dependent.</a> Acta Physiologia Plantarum. 33 (6), 2103-2116 (2011).</li><li>Nachbar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal+ion+-+dehydrin+interactions+investigated+by+affinity+capillary+electrophoresis+and+computer+models.">Metal ion - dehydrin interactions investigated by affinity capillary electrophoresis and computer models.</a> Journal of Plant Physiology. 216, 219-228 (2017).</li><li>Alhazmi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+platform+to+investigate+protein-metal+ion+interactions+by+affinity+capillary+electrophoresis.">A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.</a> Journal of Pharmaceutical and Biomedical Analysis. 107, 311-317 (2015).</li><li>Redweik, S., Xu, Y., W&#228;tzig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise,+fast,+and+flexible+determination+of+protein+interactions+by+affinity+capillary+electrophoresis:+Part+1:+Performance.">Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: Part 1: Performance.</a> ELECTROPHORESIS. 33 (22), 3316-3322 (2012).</li><li>Ghosal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetic+flow+and+dispersion+in+capillary+electrophoresis.">Electrokinetic flow and dispersion in capillary electrophoresis.</a> Annual Review of Fluid Mechanics. 38 (1), 309-338 (2006).</li><li>Xuan, X., Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+study+of+Joule+heating+effects+on+electrokinetic+transportation+in+capillary+electrophoresis.">Analytical study of Joule heating effects on electrokinetic transportation in capillary electrophoresis.</a> Journal of Chromatography A. 1064 (2), 227-237 (2005).</li><li>Oledzka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+tissue+protein+separations+applying+one-+dimensional+gel+electrophoresis+and+capillary+gel+electrophoresis.">Comparative evaluation of tissue protein separations applying one- dimensional gel electrophoresis and capillary gel electrophoresis.</a> The Open Proteomics Journal. 5 (1), 17-21 (2012).</li><li>Alhazmi, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+affinity+capillary+electrophoresis+for+routine+investigations+of+protein-metal+ion+interactions.">Optimization of affinity capillary electrophoresis for routine investigations of protein-metal ion interactions.</a> Journal of Separation Science. 38 (20), 3629-3637 (2015).</li><li>Busch, M. H. A., Carels, L. B., Boelens, H. F. M., Kraak, J. C., Poppe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+methods+for+the+study+of+drug-protein+binding+in+affinity+capillary+electrophoresis.">Comparison of five methods for the study of drug-protein binding in affinity capillary electrophoresis.</a> Journal of Chromatography A. 777 (2), 311-328 (1997).</li><li>Mozafari, M., Balasupramaniam, S., Preu, L., El Deeb, S., Reiter, C. G., W&#228;tzig, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+affinity+capillary+electrophoresis+and+computational+models+for+binding+studies+of+heparinoids+with+P-selectin+and+other+proteins.">Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with P-selectin and other proteins.</a> ELECTROPHORESIS. 38 (12), 1560-1571 (2017).</li></ol>

For all CE experiments, the preparation of the capillary is a critical step. Since it is a glass tube with a small diameter, it can easily break at the spots where the coating is removed when it is being handled. The installation of the capillary into the instrument should be done very carefully.
The critical steps involved in using the ACE method mainly pertain to the experimental setup. Another critical step is finding the right sample injection parameters. The injected amount of the sample ...

Plants are more dependent on their environment than many other life forms. Since plants cannot move to other places, they have to adjust to changes in their surroundings (e.g., drought, cold and high salt concentrations). Consequently, higher plants developed specialized stress proteins like dehydrins, which fulfill manifold tasks to reduce cell stress related to high salinity. These proteins bind water and ions inside cells, reduce oxidative stress by binding Cu2+-ions, and interact with phospholipids as well as the cytoskeletons. Moreover, binding Zn2+-ions allows these proteins to act as transcription factors. Their ability to bind Ca2+-ions after phosphorylation has also been reported1.
The multifunctional behavior of these proteins is related to the absence of hydrophobic amino acid residues. Consequently, they lack any hydrophobic interactions inside the peptide chain and also a constrained structure. However, because these proteins lack a restrictive structure, they can occupy different conformers under the same conditions. Therefore, they can be described best as an ensemble of structures rather than as a single conformation. Proteins with these properties are known as intrinsically disordered proteins (IDPs) and are a widely used concept for stress proteins and crosstalk between different pathways in eukaryotic cells2.
One of these stress-related IDPs is AtHIRD11. It is one of Arabidopsis thaliana&#39;s most highly drought-expressed IDPs. Hence, the different conformers can be separated by their effective radius to charge ratio, and capillary electrophoresis (CE) has been used for further investigations. Previous ACE experiments demonstrated the interactions between AtHIRD11 and transition metal ions such as Cu2+-, Zn2+-, Co2+, and Ni2+-ions. The detailed results can be found in Hara et al.3 and Nachbar et al.4.
The ACE method that will be used here is based on our earlier published works6. However, the addition of the EOF marker acetanilide to the protein sample is not suitable. AtHIRD11 shows broad peak patterns, and adding the EOF marker to the sample would disguise two peaks. Therefore, the marker is used in a separate run. Before the binding behavior is examined, it is confirmed that the peaks found during previous experiments are from different conformers. Thus, CGE is used to distinguish between the protein conformers, the post-translational modified protein, and impurities, such as fragments of AtHIRD11, by their different masses. Subsequently, the characterized AtHIRD11 sample&#39;s binding behavior towards various different metal ions is investigated.
The purpose of this article is to describe an experimental setup to distinguish between an IDP and other components of a sample in order to evaluate the differences in the binding behavior of different conformers.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">AtHIRD11 sample</td>
			<td style="width:111px;">Shizuoka University (Group Prof. M. Hara)</td>
			<td style="width:119px;">-</td>
			<td style="width:192px;">Dehydrin Protein from Arabidopsis thaliana expressed in Escherichia coli</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Barefused silica capillary</td>
			<td style="width:111px;">Polymicro Technologies (Phoenix, USA)</td>
			<td style="width:119px;">106815-0017</td>
			<td style="width:192px;">TSP050375, 50 &#956;m inner diameter, 363 &#956;m outer diameter, polyimide coating</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent 1600A</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td style="width:119px;">comercially not available anymore</td>
			<td style="width:192px;">Capillary electrophoresis instrument; Agilent 7100 CE can be used instead</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent 7100 CE</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td style="width:119px;">G7100A</td>
			<td style="width:192px;">Capillary electrophoresis instrument</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Injekt 2 mL</td>
			<td style="width:111px;">B. Braun (Melsungen, Germany)</td>
			<td style="width:119px;">4606051V</td>
			<td style="width:192px;">Syringe for filtration</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Rotilabo-syringe filters</td>
			<td style="width:111px;">Carl Roth GmbH + Co. KG (Karlsruhe, Germany)</td>
			<td style="width:119px;">KY62.1</td>
			<td style="width:192px;">PVDF membrane filter for solution filtration</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eppendorf Research plus 10 &#956;L</td>
			<td style="width:111px;">Eppendorf (Wesseling-Berzdorf, Germany)</td>
			<td style="width:119px;">3121 000.023</td>
			<td style="width:192px;">Micro pipette for sample handling</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Eppendorf Research plus 10 &#956;L</td>
			<td style="width:111px;">Eppendorf (Wesseling-Berzdorf, Germany)</td>
			<td style="width:119px;">3121 000.120</td>
			<td style="width:192px;">Micro pipette for handling the ligand solutions</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bulb pipette 10 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 338 08</td>
			<td style="width:192px;">Preparing theNaOH solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bulb pipette 25 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 338 14</td>
			<td style="width:192px;">Preparing the ligand solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Duran glas volumetric flask 25 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 671 1457</td>
			<td style="width:192px;">Preparing the ligand stock solution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Duran glas volumetric flask 10 mL</td>
			<td style="width:111px;">Duran Group GmbH(Mainz, Germany)</td>
			<td style="width:119px;">24 671 1054</td>
			<td style="width:192px;">Preparing the ligand stock solution</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Proteome Lab SDS MW Gel Buffer</td>
			<td style="width:111px;">Beckman Coulter (Brea, USA)</td>
			<td style="width:119px;">comercially not available anymore</td>
			<td style="width:192px;">Separation during capillary gel electrophoresis / Alternative SDS buffer: CE-SDS run buffer from Bio-Rad Laboratories (M&#252;nchen, Germany) Catalog Number: 1485032&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Acetanilide</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">397229-5G</td>
			<td style="width:192px;">Electroosmotic flow marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Manganese(II) chloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">13217</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">431788-100G</td>
			<td style="width:192px;">Rinsing ingredient</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bariumchloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">202738-5G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sodium dodecyl sulfate</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">71729-100G</td>
			<td style="width:192px;">Solublizing protein for capillary gel electrophoresis</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nickel(II) chloride hexahydrate</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">654507-5G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Selenium(IV) chloride</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">323527-10G</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-amino-2-hydroxy-methylpropane-1.3-diol (Tris)</td>
			<td style="width:111px;">Sigma-Aldrich (Steinheim, Germany)</td>
			<td style="width:119px;">252859-100G</td>
			<td style="width:192px;">Buffer ingredient</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Zinc(II) chloride</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1088160250</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Strontium nitrate</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1078720250</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Calcium chloride dihydrate</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1371015000</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">37% hydrochloric acid</td>
			<td style="width:111px;">Merck Millipore ( Darmstadt, Germany)</td>
			<td style="width:119px;">1003171000</td>
			<td style="width:192px;">Adjusting pH</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Copper(II) chloride dihydrate</td>
			<td style="width:111px;">Riedel-de Ha&#235;n (Seelze, Germany)</td>
			<td style="width:119px;">31286</td>
			<td style="width:192px;">Ligand</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sonorex Longlife RK 1028 CH 45L</td>
			<td style="width:111px;">Allpax (Papenburg, Germany)</td>
			<td style="width:119px;">10000084;0</td>
			<td style="width:192px;">Ultrasonic bath</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Agilent ChemStation Rev. 8.04.03-SP1</td>
			<td style="width:111px;">Agilent Technologies (Waldbronn, Germany)</td>
			<td>G2070-91126</td>
			<td style="width:192px;">Software packages to operate the CE instruments, acquisite data and evaluate it</td>
		</tr>
	</tbody>
</table>

analysis of athird11 intrinsic disorder and binding towards metal ions by capillary gel electrophoresis and affinity capillary electrophoresis

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve classes of intrinsically disordered proteins (IDPs) to reduce oxidative and osmotic stress. This article uses a combination of capillary gel electrophoresis (CGE) and mobility shift affinity electrophoresis (ACE) in order to describe the binding behavior of different conformers of the IDP AtHIRD11 from Arabidopsis thaliana. CGE is used to confirm the purity of AtHIRD11 and to exclude fragments, posttranslational modifications, and other impurities as reasons for complex peak patterns. In this part of the experiment, the different sample components are separated by a viscous gel inside a capillary by their different masses and detected with a diode array detector. Afterward, the binding behavior of the sample towards various metal ions is investigated by ACE. In this case, the ligand is added to the buffer solution and the shift in migration time is measured in order to determine whether a binding event has occurred or not. One of the advantages of using the combination of CGE and ACE to determine the binding behavior of an IDP is the possibility to automate the gel electrophoresis and the binding assay. Furthermore, CGE shows a lower limit of detection than the classical gel electrophoresis and ACE is able to determine the manner of binding a ligand in a fast manner. In addition, ACE can also be applied to other charged species than metal ions. However, the use of this method for binding experiments is limited in its ability to determine the number of binding sites. Nevertheless, the combination of CGE and ACE can be adapted for characterizing the binding behavior of any protein sample towards numerous charged ligands.

Figure 1 shows the electropherogram for the AtHIRD11 sample obtained during the CGE experiments. The peptide size increases from left to right. Peak number 4 has the largest mass and indicates the intact protein. The smaller peaks 2 and 3 represent smaller impurities (e.g., degradation products). The first peak and the inconsistency of the baseline before could also be reproduced without the protein sample. Therefore, it is related to the SDS gel its...

Watch this Scientific Journal Video about Analysis of AtHIRD11 Intrinsic Disorder and Binding Towards Metal Ions by Capillary Gel Electrophoresis and Affinity Capillary Electrophoresis at JoVE.com

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

This protocol combines the characterization of a protein sample by capillary gel electrophoresis and a fast-binding screening for charged ligands by affinity capillary electrophoresis. It is recommended for proteins with a flexible structure, such as intrinsically disordered proteins, to determine any differences in binding for different conformers.

Plants are strongly dependent on their environment. In order to adjust to stressful changes (e.g., drought and high salinity), higher plants evolve ...

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