Name: demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay
Uploaded: 2020-09-10T15:30:00
Description: Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Dette arbejde blev støttet af bevilling nr. 2016/23/D/NZ3/01314 fra National Science Centre (NCN), Krakow, Polen.

1. Generering af ekspressionsplasmider
<ol><li>Undersøg membrantopologi af de proteiner, der er af interesse, ved hjælp af et topologiforudsigelsesværktøj.</li>
	<li>Design kloningsstrategien, så de større og mindre fragmenter vil vende mod cytoplasmaet, når fusionsproteinerne er blevet indsat i Golgi-membraner. Hvis både N- og C-termini af de proteiner af interesse, som i det tilfælde, der præsenteres her, er cytoplasmatisk orienterede, mærke proteinerne på otte mulige måder (se figur...

<ol>
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A., Coincon, M., Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+delivery+of+activated+sialic+acid+into+Golgi+for+sialyation.">Structural basis for the delivery of activated sialic acid into Golgi for sialyation.</a> Nature Structural and Molecular Biology. 26 (6), 415-423 (2019).</li><li>Parker, J. L., Corey, R. A., Stansfeld, P. J., Newstead, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+substrate+specificity+and+regulation+of+nucleotide+sugar+transporters+in+the+lipid+bilayer.">Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer.</a> Nature Communications. 10 (1), 4657 (2019).</li><li>Wiertelak, W., Sosicka, P., Olczak, M., Maszczak-Seneczko, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+homologous+and+heterologous+interactions+between+UDP-galactose+transporter+and+beta-1,4-galactosyltransferase+1+using+NanoBiT.">Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT.</a> Analytical Biochemistry. 593, 113599 (2020).</li><li>Hong, K., Ma, D., Beverley, S. M., Turco, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Leishmania+GDP-mannose+transporter+is+an+autonomous,+multi-specific,+hexameric+complex+of+LPG2+subunits.">The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits.</a> Biochemistry. 39 (8), 2013-2022 (2000).</li><li>Sosicka, P., 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=An+insight+into+the+orphan+nucleotide+sugar+transporter+SLC35A4.">An insight into the orphan nucleotide sugar transporter SLC35A4.</a> Biochimica et Biophysica Acta. Molecular Cell Research. 1864 (5), 825-838 (2017).</li><li>Sprong, H., 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=Association+of+the+Golgi+UDP-galactose+transporter+with+UDP-galactose:ceramide+galactosyltransferase+allows+UDP-galactose+import+in+the+endoplasmic+reticulum.">Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum.</a> Molecular Biology of the Cell. 14 (8), 3482-3493 (2003).</li><li>Maszczak-Seneczko, D., 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=UDP-galactose+(SLC35A2)+and+UDP-N-acetylglucosamine+(SLC35A3)+Transporters+Form+Glycosylation-related+Complexes+with+Mannoside+Acetylglucosaminyltransferases+(Mgats).">UDP-galactose (SLC35A2) and UDP-N-acetylglucosamine (SLC35A3) Transporters Form Glycosylation-related Complexes with Mannoside Acetylglucosaminyltransferases (Mgats).</a> Journal of Biological Chemistry. 290 (25), 15475-15486 (2015).</li><li>Khoder-Agha, F., 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=N-acetylglucosaminyltransferases+and+nucleotide+sugar+transporters+form+multi-enzyme-multi-transporter+assemblies+in+golgi+membranes+in+vivo.">N-acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme-multi-transporter assemblies in golgi membranes in vivo.</a> Cellular and Molecular Life Sciences. 76 (9), 1821-1832 (2019).</li><li>Dixon, A. S., 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=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Tung, J. K., Berglund, K., Gutekunst, C. -. A., Hochgeschwender, U., Gross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+live+cells+and+animals.">Bioluminescence imaging in live cells and animals.</a> Neurophotonics. 3 (2), 025001 (2016).</li><li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Molecular Cell. 9 (4), 789-798 (2002).</li><li>Inoue, 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=Illuminating+G-Protein-Coupling+Selectivity+of+GPCRs.">Illuminating G-Protein-Coupling Selectivity of GPCRs.</a> Cell. 177 (7), 1933-1947 (2019).</li><li>White, C. W., Caspar, B., Vanyai, H. K., Pfleger, K. D. G., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Mediated+Protein+Tagging+with+Nanoluciferase+to+Investigate+Native+Chemokine+Receptor+Function+and+Conformational+Changes.">CRISPR-Mediated Protein Tagging with Nanoluciferase to Investigate Native Chemokine Receptor Function and Conformational Changes.</a> Cell Chemical Biology. 27, 499-510 (2020).</li><li>Akinjiyan, F. 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+Novel+Luminescence-Based+High-Throuput+Approach+for+Cellular+Resolution+of+Protein+Ubiquitination+using+Tandem+Ubiquitin+Binding+Entities+(TUBEs).">A Novel Luminescence-Based High-Throuput Approach for Cellular Resolution of Protein Ubiquitination using Tandem Ubiquitin Binding Entities (TUBEs).</a> SLAS Discovery. 25 (4), 350-360 (2020).</li><li>Soave, M., Kellam, B., Woolard, J., Briddson, S. J., Hill, S. 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Her giver vi en detaljeret protokol, der muliggør demonstration af heterologe komplekser dannet mellem Golgi-residente type III-membranproteiner, såsom NST'er, ved hjælp af det delte luciferase-komplementeringsassay. Den foreslåede tilgang til dataanalyse og -fortolkning indebærer at relatere den luminescens, der er opnået for proteinkombinationen af interesse, til den luminescens, der er opnået for den tilsvarende kontrolkombination, som består af et af de proteiner af interesse, der er smeltet sammen med det st...

Dette manuskript beskriver en trinvis protokol til kontrol af tilstedeværelsen af heterologe interaktioner mellem Golgi-resident type III-membranproteiner i forbigående transfekterede humane celler ved hjælp af den seneste variant af split luciferase-komplementeringsassayet. Proceduren er blevet grundigst testet mod nukleotidsukkertransportører (NST'er), men vi var også i stand til at opnå positive resultater for andre Golgi-hjemmehørende type III-membranproteiner, hvis N- og / eller C-termini står over for cytoplasmaet.
Vores forskningsgruppe undersøger NST'ernes rolle i glykosylering af makromolekyler. NST'er er Golgi- og/eller ER-resident type III-membranproteiner med N- og C-termini vendt mod den cytoplasmatiske side af den organellare membran1. NST'er menes at bære nukleotidaktiverede sukkerarter over organelmembraner for at levere glycosyltransferaser med deres substrater. NST'er danner dimerer og/eller højere oligomerer2,3,4,5,6,7,8,9,10. Desuden er heterologe interaktioner mellem forskellige NST'er også blevet rapporteret6,11. NST'er blev også påvist at danne komplekser med funktionelt beslægtede glykosyleringsenzymer12,13,14. Vi søgte efter et alternativ til den i øjeblikket anvendte teknik, fluorescens levetidsbilleddannelse (FLIM) -baseret FRET-tilgang, til at studere interaktioner mellem NST'er og funktionelt relaterede Golgi-residente proteiner, så vi besluttede at teste split luciferase-komplementeringsassay. Det gjorde det muligt for os at identificere en ny interaktion mellem en NST og et funktionelt relateret glykosyleringsenzym9.
Den seneste ændring af det opdelte luciferase-komplementeringsassay, NanoBiT, anvendes i den protokol, der præsenteres her15. Det er afhængig af rekonstitutionen af luciferaseenzymet (f.eks. NanoLuc) fra de to fragmenter - den store, betegnet som stor BiT eller LgBiT, et 17,6 kDa-protein, og den lille, der kun består af 11 aminosyrer, betegnet som lille BiT eller SmBiT. De to proteiner af interesse smeltes sammen med de komplementære fragmenter og udtrykkes forbigående i en menneskelig cellelinje. Hvis de to fusionsproteiner interagerer, produceres en luminescens in situ ved tilsætning af et cellegennemtrængeligt substrat. Disse to fragmenter er blevet optimeret, så de forbinder med minimal affinitet, medmindre de bringes sammen af en interaktion mellem de proteiner af interesse, de er smeltet sammen med.
Generelt har bioluminescensbaserede metoder nogle fordele i forhold til dem, der er baseret på fluorescens. Bioluminescerende signaler har et højere signal-støj-forhold, fordi baggrundsluminescensen er ubetydelig sammenlignet med det luciferase-afledte signal16. I modsætning hertil lider fluorescensbaserede tilgange normalt af en relativt høj baggrund forårsaget af fænomenet autofluorescens. Desuden er bioluminescens mindre skadelig for de analyserede celler end fluorescens, da der i det førstnævnte tilfælde ikke er behov for at ophidse prøven. Af disse grunde udkonkurrerer bioluminescerende tilgange til undersøgelse af PPI in vivo de almindeligt anvendte fluorescerende metoder som Förster Resonance Energy Transfer (FRET) eller Bimolekylær fluorescenskomplementering (BiFC).
Vores protokol er afhængig af henvisende luminescens opnået for proteinkombinationen af interesse for luminescens opnået for kontrolkombinationen. Sidstnævnte omfatter et af de testede proteiner, der er smeltet sammen med et større fragment og et kontrolprotein (f.eks. HaloTag), smeltet sammen med et mindre fragment. Sidstnævnte er et protein af bakteriel oprindelse, der ikke forventes at interagere med nogen af pattedyrproteiner. Brug af dette protein som kontrol udgør begrænsninger for topologien af de Golgi-hjemmehørende par af proteiner, der skal analyseres. Da dette protein i pattedyrceller syntetiseres i cytoplasmaet, bør begge proteiner af interesse have mindst en cytoplasmatisk hale.
Denne tilgang kan være særlig nyttig til indledende screening af PPI. Det kan blive den valgte metode, når fusionsproteinerne af interesse udtrykkes på niveauer, der simpelthen er utilstrækkelige til, at andre tilgange kan anvendes. Tilsvarende kan split luciferase-komplementeringsassay være den bedste løsning, hvis proteinerne af interesse udtrykkes på høje niveauer, men dette påvirker deres subcellulære lokalisering negativt eller er kendt for at tvinge ikke-specifikke interaktioner. Da det mindre fragment kun har 11 aminosyrer, kan det splittede luciferasekomplementeringsassay anvendes, når det er umuligt at bruge større tags. Endelig kan det anvendes til yderligere at bekræfte data opnået ved hjælp af andre teknikker, som i det tilfælde, der præsenteres her.

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent mammalian cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td>
			<td>Corning</td>
			<td>356651</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE HD Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td>
			<td>Promega</td>
			<td>GM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium dedicated to the cell line used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT MCS Starter System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td>
		</tr>
		<tr>
			<td>Nano-Glo Live Cell Assay System</td>
			<td>Promega</td>
			<td>N2011</td>
			<td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium, no phenol red</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Oribital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for data analysis (e.g. GraphPad Prism)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

Målet med denne protokol er at undersøge anvendeligheden af den seneste variant af split luciferase-komplementering til påvisning af heterologe komplekser dannet af nukleotidsukkertransportører (NST'er). Disse ER- og Golgi-residente multitransmembranproteiner bærer de cytoplasmatisk syntetiserede nukleotidsukker over organelmembraner for at levere enzymer, der medierer glykosylering med deres substrater. NST'er findes som dimerer og/eller højere oligomerer. Heterologe interaktioner mellem forskellige NST'er er også blevet rapporteret. For at kontrollere, om teknikken er egnet til at studere fænomenet NST-heteromerisering, testede vi den mod en kombination af de to Golgi-residente NST'er, der tidligere har vist sig at associere på flere andre måder. Luciferase-komplementeringsassayet ser ud til at være særligt velegnet til at studere interaktioner mellem Golgi-residente membranproteiner, da det ikke kræver høje ekspressionsniveauer, hvilket ofte udløser proteinfejllokalisering og øger risikoen for falske positiver.

For at opnå de mest pålidelige data i denne tilgang bør alle mulige kombinationer testes (jf . figur 1). Parallelt hertil bør positive og negative kontroller medtages. Den positive kontrol skal bestå af de to proteiner, der vides at interagere, hvoraf det ene er smeltet sammen med det større fragment, og det andet er smeltet sammen med det mindre fragment. Den negative kontrol bør ideelt set bestå af de to ikke-interagerende type III-membranproteiner, der er mærket på samme måde. ...

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Protokollen, der præsenteres her, har til formål at påvise forekomsten af heterologe interaktioner mellem Golgi-hjemmehørende type III-membranproteiner med cytoplasmatisk eksponerede N- og/eller C-termini i levende pattedyrceller ved anvendelse af den seneste variant af det splittede luciferasekomplementeringsassay.

Demonstration af heterologe komplekser dannet af Golgi-Resident Type III membranproteiner ved anvendelse af Split Luciferase Complementation Assay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

Our protocol allows to identify and analyze interactions between multi transmembrane proteins of the endoplasmic reticulum and golgi complex in living cells at low expression levels. The main advantage of this technique is it's to special sensitivity, which allows to avoid high over expression levels that are often detrimental to cellular health and may disturb protein subcellular localization. Harvest the adherent HEK293 T-cell culture by trypsinization and re-suspend the cells in a dedicated complete growth medium.Plate the cells onto a clear bottom, white side 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls, including replicates. Then, culture the cells overnight in standard conditions.On the next day, transfect the cells with the desired combinations of expression plasmids. Dilute the plasmids in a serum-free medium to 6.25 nanograms per microliter for each construct and add the lipid-based transfection reagent. Add an appropriate lipid to DNA ratio.Incubate the plasmids according to manufacturer's instruction. Then, add eight microliters of the lipid DNA mixture to the designated wells. Mix the content of the plate with gentle rotation and culture the cells for 20 24 hours in standard conditions.On the next day, replace the conditioned medium in each well with 100 microliters of a serum-free medium, making sure that the cells do not detach upon medium exchange. Just before measuring luminescence, mix one volume of furimazine with 19 volumes of a dilution buffer. Add 25 microliters of the furimazine working solution into each well, and gently mix the plate by hand or with a orbital shaker.Insert the plate into a luminescence micro plate reader and equilibrate the plate at 37 degrees Celsius for 10 15 minutes. Select the wells to be analyzed and read luminescence with an integration time of 0.3 seconds. Continue to monitor luminescence for up to two hours when required.This protocol was tested on two nucleotide sugar transporters, SLC35A2 and SLC35A3, which are golgi-resident type three membrane proteins with N and C termini facing the cytoplasm. There were eight possible tagging options and the resulting fusion proteins could be set together in eight possible ways. Mean relative luminescence units or RLU values corresponding to all the tested and control combinations are shown here.The RLU values obtained for three of the combinations were significantly higher than those obtained for the corresponding control combination. To obtain ratio values for the combinations of interest, the RLU values were divided by the RLU values obtained for the respective controls. Ratios between 10 and 1000 are highly indicative of specific interactions.There were two such combinations supporting the idea that SLC35A2 and SLC35A3 interact. To confirm this data, HA tagged SLC35A2 or SLC35A3 were co-expressed with the combination that resulted in the highest relative luminescence. Varying amounts of the plasmids and coding HA tagged NST variants were used for co-transfection, resulting in a statistically significant dose dependent decrease in RLU values.The most critical thing is to tackle your protein self interest in all possible ways and to assay all possible combination, as some tagging orientation may produce false negative results. Once the best performing combination has been selected, the dynamics of the corresponding interaction can be monitored to, for example identify agents or conditions that disrupt your complex of interest over time.

Research

JoVE Journal

Biology

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

High-throughput krystallisering af membranproteiner Brug af Lipidic Bicelle Metode

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Anvendelse af en robot til Højkapacitetsforskning Krystallisation af membranproteiner i Lipidiske mesofaser

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Høst og Cryo-køling Krystaller af membranproteiner dyrket i Lipidiske mesofaser for Structure Bestemmelse ved makromolekylær krystallografi

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

High-throughput Funktionel Screening ved hjælp af en hjemmelavet Dual-glød Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...