Name: capturing the interaction kinetics of an ion channel protein with small molecules by the bio-layer interferometry assay
Uploaded: 2018-03-07T13:30:00
Description: Watch this Scientific Journal Video about Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay at JoVE.com

Dette arbejde blev støttet af Bio-ID Center og SJTU tværfaglig forskningsfond i medicin og teknik (YG2016QN66), National Natural Science Foundation of China (31271217) og nationale grundlæggende forskning Program af Kina (2014CB910304).

Bemærk: Linjen HEK-293T celle løbende udtrykker FLAG-markeret hEAG1 kanal protein er konstrueret af transfecting en pCDH lentiviral plasmid som indeholder DNA sekvens af hEAG1 med et FLAG på den distale C-terminus HEK-293T celler efterfulgt af den puromycin-resistente udvalg som tidligere beskrevet15.
1. affinitet rensning af FLAG-markeret hEAG1 kanal Protein fra HEK-293T celler
<ol><li>Tø celler stabilt at udtrykke hEAG1 kanaler fra flydende kvælstof til 37 ° C...

<ol>
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Membran Ionkanaler er blevet bekræftet som de primære terapeutiske mål på over 13% af aktuelt kendte lægemidler til behandling af en lang række sygdomme, herunder hjerte-kar- og neurologiske lidelser18. Patch-klemme optagelse, har den gyldne standard for måling af de funktionelle Ionkanaler med små molekyler, været meget anvendt for ion-kanal ligander screening. Dog kan ikke sådan elektrofysiologiske metoder påvise om de små molekyler bindes til kanal direkte eller ikke<sup class="xref...

Målretning celle overflade-tilgængelige ion-kanal proteiner med små molekyler giver et enormt potentiale for ligand screening og biologiske drug discovery1,2,3. Således, en passende værktøj er nødvendig for at studere samspillet mellem ion-kanal og små molekyler og deres tilsvarende funktion. Patch-clamp optagelse har vist sig for at være en enestående og uerstattelige teknik i ion-kanal funktionelle analyse. Dog fastslå, om de små molekyler direkte målrette Ionkanaler kræver andre teknologier. Traditionelt, blev radioaktive ligand bindende assay brugt til at observere kinetik af binding mellem lille molekyle og dens mål ion-kanal protein. Brugen af denne teknik er dog begrænset på grund af dens krav i radioaktive mærkning og registrering. Desuden forhindrer de forudsætning skridt til at mærke de små ligand i undersøgelsen, dens bruger i mange typer af Ionkanaler uden kendte specifikke ligand. Nogle etiket-fri teknikker såsom NMR spektroskopi, røntgen diffraktion, individuel thermophoresis (MST)4 og overflade plasmon resonans (SPR) har været brugt til at måle de protein-lille molekyle interaktioner. Men disse typer af assays normalt ikke kan give tilstrækkelige oplysninger på grund af vanskeligheder ved at få fuld længde protein, lav opløsning af dynamics, lav overførselshastighed og høje omkostninger5. I modsætning til disse teknikker fremstår bio-lag interferometri (BLI) som en roman etiket-fri metode til at overvinde disse ulemper til påvisning af protein-lille molekyle interaktioner ved immobilisere et bittesmå mængder af protein prøve på overflader biosensor og måling af den optiske skiftende signaler6,7. Som en lovende biosensor platform, BLI teknik er allerede udført for at observere samspillet mellem små molekyler med naturlig vand opløselige proteiner som et humant monoklonalt antistof CR80208 og den detaljerede analyseprocedure er blevet rapporteret i en tidligere artikel9. Selvom nøglerolle ion-kanal protein for nye terapeutiske mål opdagelse er blevet anerkendt, ion-kanal protein-lille molekyle interaktion analysen baseret på BLI ikke er blevet beskrevet.
Menneskelige Ether à go-go kanaler (hEAG1) er udtrykt i forskellige typer af kræftceller og centrale nervesystem, hvilket gør kanal a potentielle terapeutiske mål for mange kræftformer og neuronale forstyrrelser10,11, 12,13,14. Den elektrofysiologiske undersøgelse i vores lab har bekræftet den hæmmende effekt af phosphatidylinositol 4, 5-bisfosfat (PIP2) på hEAG1 kanal15. Baseret på vores resultater, test PIP2 direkte interaktion med hEAG1 ved hjælp af BLI teknik kan være et forbillede for andre typer af ion-kanal protein-lille molekyle sammensat interaktion især for de kanaler, der mangler specifikke ligander. Henhold til instruktionerne BLI assay, vi forberedte biotinylated hEAG1 proteiner og immobiliseret dem på overfladen af streptavidin (SA) biosensor tips efterfulgt af interaktion dem til PIP2 løsninger at observere deres direkte bindende mellem den protein og lipid. Efter udlæg i PIP2 hEAG1 protein coated overflade, tykkelse af lag på overfladen øger, som direkte korrelerer den spektrale Skift og kan måles i real-time16. Bindende kinetik kan bestemmes på grund af en positiv forskydning i foreningen trin og en negativ forskydning i dissociation trin. Ifølge dette princip vi renset funktionelle hEAG1 ion-kanal protein fra HEK-239T stabil udtryk system ved hjælp af affinitet rensning metode til at opretholde den in vitro- funktionelle tilstand, derefter målt kinetik af bindingen af forskellige koncentration PIP2, og gav en semblable kinetiske data som observeret i elektrofysiologiske målinger15. Tæt korrespondancen mellem resultaterne fra BLI og elektrofysiologiske målinger viser for første gang BLI egnethed som et relevant analytisk værktøj for ion-kanal membran protein-lille molekyle interaktion.

<table>
	<tbody>
		<tr>
			<td>DMEM/High Glucose Medium</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (1x)</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>1697550</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430599</td>
			<td>150 mm X 25 mm</td>
		</tr>
		<tr>
			<td>Nonidet P-40 Substitute</td>
			<td>Amresco</td>
			<td>E109</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610476</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BBI Life Sciences</td>
			<td>A610440</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>BBI Life Sciences</td>
			<td>A600332</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene-20-Sorbitan Monolaurate</td>
			<td>BBI Life Sciences</td>
			<td>A600560</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Sigma</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>3x FLAG peptide</td>
			<td>Sigma</td>
			<td>F4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Octet-RED96</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>30-5048</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Analysis software</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>Version 7.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosensor/Streptavidin</td>
			<td>Pall/Fort&#233;Bio</td>
			<td>18-5019</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtiter plate</td>
			<td>Greiner Bio-one</td>
			<td>655209</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-NHS-LC-LC-Biotin</td>
			<td>ThermoFisher</td>
			<td>21338</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugal Machine</td>
			<td>ThermoFisher</td>
			<td>75004250</td>
			<td></td>
		</tr>
		<tr>
			<td>PageRuler Prestained Protein Ladder</td>
			<td>ThermoScientific</td>
			<td>318120</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrafiltration device</td>
			<td>MILLIPORE</td>
			<td>UFC503008</td>
			<td>NMWL of 30 kDa</td>
		</tr>
		<tr>
			<td>phosphatidylinositol 4, 5-bisphosphate (PIP2)</td>
			<td>Sigma</td>
			<td>P9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal ANTI-FLAG M2 antibody</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>goat anti-mouse HRP-conjugated secondary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2005</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr>
			<td>Enhanced BCA Protein Assay Kit</td>
			<td>Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>04693159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Imager 600 Imaging System</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Western blot system</td>
			<td>BIO-RAD</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing the interaction kinetics of an ion channel protein with small molecules by the bio-layer interferometry assay

Bio-lag interferometri (BLI) assay er et værdifuldt redskab til måling af protein-protein og protein-lille molekyle interaktioner. Her, vi først beskrive anvendelsen af denne roman etiket-gratis teknik til at studere samspillet mellem menneskelige EAG1 (hEAG1) kanal proteiner med lille molekyle PIP2. hEAG1 kanal er blevet anerkendt som potentielle terapeutiske mål på grund af dets afvigende overekspression i kræft og få gevinst af funktion mutationer involveret i nogle typer af neurologiske sygdomme. Vi renset hEAG1 kanal proteiner fra et pattedyr stabil ekspressionssystem og målt interaktion med PIP2 af BLI. Vellykket måling af kinetik af binding mellem hEAG1 protein og PIP2 viser, at BLI assay er en potentiel høj overførselshastighed tilgang anvendes til nye små-molekyle ligand screening i ion-kanal farmakologi.

Vi renset FLAG fusion hEAG1 kanal protein fra HEK-293T celler stabilt overexpressed hEAG1. Funktionen af denne fusion protein er blevet påvist ved hjælp af metoden patch-clamp og kvalitet og specificitet af oprenset protein er bekræftet af vestlige skamplet (figur 1). Renset kanal protein er biotinylated til at udføre en interaktion assay med lipider (PIP2) ved hjælp af real-time BLI analysen. BLI bindende assay konfiguration er vist i <strong...

Watch this Scientific Journal Video about Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay at JoVE.com

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

Protokol her beskriver renset hEAG1 ion-kanal protein interaktioner med lille molekyle lipid ligand phosphatidylinositol 4, 5-bisfosfat (PIP2). Målingen viser, at BLI kunne være en potentiel metode til romanen små-molekyle ion-kanal ligand screening.

Indfange interaktion kinetik af en Ion-kanal Protein med små molekyler af Bio-lag interferometri Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Research

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Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Fremgangsmåde til at identificere småmolekylære inhibitorer af protein-protein-vekselvirkning mellem HCN1 og TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Visualisering af protein-protein interaktion i nukleare og Cytoplasmiske Brøker af Co-immunopræcipitation og<em&gt; In Situ</em&gt; Proximity ligeringsassayet

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

Kraft spektroskopi af enkelt proteinmolekyler ved hjælp af en Atomic Force mikroskop

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Measurement of Ion Concentration in the Unstirred Boundary Layer with Open Patch-Clamp Pipette: Implications in Control of Ion Channels by Fluid Flow

Måling af Ion koncentration i Unstirred grænselag med åben Patch-Clamp Pipette: konsekvenser i kontrol af Ionkanaler af væske Flow

Fluid flow is an important environmental stimulus that controls many physiological and pathological processes, such as fluid flow-induced vasodilation. ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

Ionbytning kromatografi (IEX) koblet til multi-vinkel lysspredning (MALS) for Protein adskillelse og karakterisering

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

En semi-High-gennemløb tilpasning af NADH-koblede ATPase assay til screening små molekyle hæmmere

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

En In Vitro Single-Molecule Imaging Assay til analyse af cap-afhængige oversættelse kinetik

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Derivatization of Protein Crystals with I3C using Random Microseed Matrix Screening

Derivatization af Protein Krystaller med I3C ved hjælp af Random Microseed Matrix Screening

Protein structure elucidation using X-ray crystallography requires both high quality diffracting crystals and computational solution of the diffraction ...

Microcrystal Electron Diffraction of Small Molecules

Mikrokrystalelektrondiffraktion af små molekyler

A detailed protocol for preparing small molecule samples for microcrystal electron diffraction (MicroED) experiments is described. MicroED has been ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

Identifikation af bindingsproteiner fra små ligands med Ligand Assays differentierede radiale kapillærhandling (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...