Name: in vitro characterization of histone chaperones using analytical, pull-down and chaperoning assays
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De ekstramurale tilskud til Dileep Vasudevan fra Science and Engineering Research Board, Indiens regering [CRG/2018/000695/PS] og Institut for Bioteknologi, Ministeriet for Videnskab og Teknologi, Indiens regering [BT/INF/22/SP33046/2019] samt den intramurale støtte fra Institute of Life Sciences, Bhubaneswar anerkendes i høj grad. Vi takker fru Sudeshna Sen og fru Annapurna Sahoo for deres hjælp med histonpræparation. Diskussionerne med vores kolleger Dr. Chinmayee Mohapatra, Mr. Manas Kumar Jagdev og Dr. Shaikh Nausad Hossain anerkendes også.

1. Analytisk størrelsesekskluderingskromatografi til belysning af histonchaperoners oligomere status og stabilitet
<ol><li>Analyse af histon chaperoners oligomere status<ol><li>Ækvivalent mellem en 24 ml analytisk størrelsesekskluderende kromatografi (SEC) kolonne med 1,2 kolonnevolumen (CV), dvs. 28,8 ml afgasset SEC-buffer [20 mM Tris-HCl (pH 7,5), 300 mM NaCl og 1 mM β-mercaptoethanol (β-ME)] ved 4 °C (se materialetabel). BEMÆRK: Kolonnetype, buffersammensætning og buffer...

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H., Roemer, S. C., Port, A. M., Churchill, M. E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAF-1-induced+oligomerization+of+histones+H3/H4+and+mutually+exclusive+interactions+with+Asf1+guide+H3/H4+transitions+among+histone+chaperones+and+DNA.">CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.</a> Nucleic Acids Research. 45 (16), 9809 (2017).</li><li>Bowman, 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=The+histone+chaperones+Vps75+and+Nap1+form+ring-like,+tetrameric+structures+in+solution.">The histone chaperones Vps75 and Nap1 form ring-like, tetrameric structures in solution.</a> Nucleic Acids Research. 42 (9), 6038-6051 (2014).</li><li>Newman, E. 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Dette arbejde demonstrerer og validerer et omfattende sæt protokoller til biokemisk og biofysisk karakterisering af en formodet histon chaperone. Heri blev rekombinant udtrykte og oprensede NAP-familieproteiner, AtNRP1 og AtNRP2, og det N-terminale nukleoplasmindomæne af AtFKBP53 brugt til at demonstrere protokollerne. Det samme sæt eksperimenter kunne meget vel bruges til at afgrænse de funktionelle egenskaber ved tidligere ukarakteriserede histonchaperoner fra enhver organisme.
Den førs...

Nukleosomer sammensat af DNA- og histonproteiner danner den strukturelle enhed af kromatin og regulerer flere kritiske cellulære hændelser. Nukleosomer omplaceres dynamisk og ombygges for at gøre DNA tilgængeligt for forskellige processer såsom replikation, transkription og oversættelse 1,2. Histoner, der er meget basiske, har enten tendens til at interagere med sure proteiner i det cellulære miljø eller gennemgå aggregering, hvilket fører til forskellige cellulære defekter 3,4,5. En gruppe dedikerede proteiner kaldet histon chaperoner hjælper transporten af histoner fra cytoplasmaet til kernen og forhindrer afvigende histon-DNA-aggregeringshændelser 6,7. Grundlæggende opbevarer og overfører de fleste histonkaperoner histoner til DNA ved fysiologisk ionstyrke og derved hjælper dannelsen af nukleosomer 8,9. Nogle histon chaperoner har en klar præference for histon oligomerer H2A / H2B eller H3 / H410.
Histone chaperoner er karakteriseret baseret på deres evne til at samle nukleosomer afhængige eller uafhængige af DNA-syntese11. For eksempel er kromatinsamlingsfaktor-1 (CAF-1) afhængig, mens histonregulator A (HIRA) er uafhængig af DNA-syntese12,13. Tilsvarende er nukleoplasminfamilien af histon chaperoner involveret i sædkromatindekondensation og nukleosomsamling14. Nukleosomsamlingsproteinet (NAP) familiemedlemmer letter dannelsen af nukleosomlignende strukturer in vitro og er involveret i shuttling af histoner mellem cytoplasma og kerne15. Nukleoplasminer og NAP-familieproteiner er begge funktionelle histonchaperoner, men deler ingen strukturelle træk. I det væsentlige tillader ingen enkelt strukturel funktion klassificering af et protein som en histon chaperone16. Brugen af funktionelle og biofysiske assays sammen med strukturelle undersøgelser fungerer bedst til at karakterisere histon chaperoner.
Dette arbejde beskriver biokemiske og biofysiske metoder til at karakterisere et protein som en histon chaperon, der hjælper nukleosom samling. For det første blev analytisk størrelsesekskluderingskromatografi udført for at analysere den oligomere status og stabilitet af histonchaperoner. Dernæst blev der udført et pull-down-assay for at bestemme drivkræfterne og den konkurrencemæssige karakter af histon chaperone-histon-interaktioner. Imidlertid kunne de hydrodynamiske parametre for disse interaktioner ikke beregnes nøjagtigt ved hjælp af analytisk størrelsesekskluderingskromatografi på grund af proteinets form og dets komplekser, der påvirker deres migration gennem søjlen. Derfor blev analytisk ultracentrifugering anvendt, hvilket giver makromolekylære egenskaber i opløsning, der inkluderer nøjagtig molekylvægt, interaktionens støkiometri og formen af de biologiske molekyler. Tidligere undersøgelser har i vid udstrækning brugt in vitro histon chaperoning assay til funktionelt at karakterisere histon chaperoner såsom yScS11617, DmACF18, ScRTT106p19, HsNPM120. Histone chaperoning assay blev også brugt til funktionelt at karakterisere proteinerne som histon chaperoner.

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	<tbody>
		<tr>
			<td>Acetic acid (glacial)</td>
			<td>Sigma</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>MP Biomedicals</td>
			<td>814326</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>MP Biomedicals</td>
			<td>193983</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA Pure 25M FPLC</td>
			<td>Cytiva</td>
			<td>29018226</td>
			<td>Instrument for protein purification</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>An-60Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>361964</td>
			<td>Rotor for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie brilliant blue R 250</td>
			<td>Sigma</td>
			<td>1.15444</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing (7 kDa cut-off)</td>
			<td>Thermo Fisher</td>
			<td>68700</td>
			<td>For dialysing protein samples</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>MP Biomedicals</td>
			<td>100597</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Loading Dye</td>
			<td>New England Biolabs</td>
			<td>B7025S</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>MP Biomedicals</td>
			<td>194822</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Shimadzu</td>
			<td>ATX224R</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide (EtBr)</td>
			<td>Sigma</td>
			<td>E8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Doc System</td>
			<td>Bio-Rad</td>
			<td>12009077</td>
			<td>For imaging gels after staining</td>
		</tr>
		<tr>
			<td>Horizontal gel electrophoresis apparatus</td>
			<td>Bio-Rad</td>
			<td>1704405</td>
			<td>Instrument for agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>MP Biomedicals</td>
			<td>102033</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Eppendorf</td>
			<td>Z683779</td>
			<td>For pipetting of micro-volumes</td>
		</tr>
		<tr>
			<td>Mini-PROTEAN electrophoresis system</td>
			<td>Bio-Rad</td>
			<td>1658000</td>
			<td>Instrument for SDS-PAGE</td>
		</tr>
		<tr>
			<td>N,N-methylene-bis-acrylamide</td>
			<td>MP Biomedicals</td>
			<td>800172</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano drop</td>
			<td>Thermo Fisher</td>
			<td>ND-2000</td>
			<td>For measurement of protein and DNA concentrations</td>
		</tr>
		<tr>
			<td>Ni-NTA agarose</td>
			<td>Invitrogen</td>
			<td>R901-15</td>
			<td>Resin beads for pull-down assay</td>
		</tr>
		<tr>
			<td>Optima AUC analytical ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>B86437</td>
			<td>Instrument for analytical ultracentrifugation</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>MT30130863</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol</td>
			<td>Sigma</td>
			<td>P4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>1.07393</td>
			<td></td>
		</tr>
		<tr>
			<td>pUC19</td>
			<td>Thermo Fisher</td>
			<td>SD0061</td>
			<td>Plasmid for supercoiling assay</td>
		</tr>
		<tr>
			<td>Refrigerated high-speed centrifuge</td>
			<td>Thermo Fisher</td>
			<td>75002402</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS-PAGE protein marker</td>
			<td>Bio-Rad</td>
			<td>1610317</td>
			<td></td>
		</tr>
		<tr>
			<td>SEDFIT</td>
			<td></td>
			<td></td>
			<td>Free software program for analytical ultracentrifugation data analysis</td>
		</tr>
		<tr>
			<td>SEDNTERP</td>
			<td></td>
			<td></td>
			<td>Free software program to estimate viscosity and density of buffer and partial specific volume of a protein</td>
		</tr>
		<tr>
			<td>SigmaPrep Spin Columns</td>
			<td>Sigma</td>
			<td>SC1000</td>
			<td>For pull-down assay</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Merck</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>MP Biomedicals</td>
			<td>102918</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>28990944</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>Superdex 75 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>29148721</td>
			<td>Column for analytical size-exclusion chromatography</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma</td>
			<td>1.10732</td>
			<td></td>
		</tr>
		<tr>
			<td>Topoisomerase I</td>
			<td>Inspiralis</td>
			<td>WGT102</td>
			<td>Enzyme used in plasmid supercoiling assay</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Merck</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>MP Biomedicals</td>
			<td>191450</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>N&#252;ve</td>
			<td>NB 5</td>
			<td>For heat treatment of protein samples</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol (&#946;-ME)</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro characterization of histone chaperones using analytical, pull-down and chaperoning assays

Histonproteiner associeres med DNA for at danne det eukaryote kromatin. Den grundlæggende enhed af kromatin er et nukleosom, der består af en histonoktamer bestående af to kopier af kernehistonerne H2A, H2B, H3 og H4, viklet rundt af DNA'et. Octamere består af to kopier af en H2A/H2B dimer og en enkelt kopi af en H3/H4 tetramer. De højt ladede kernehistoner er tilbøjelige til ikke-specifikke interaktioner med flere proteiner i den cellulære cytoplasma og kernen. Histone chaperoner danner en forskelligartet klasse af proteiner, der transporterer histoner fra cytoplasmaet ind i kernen og hjælper deres aflejring på DNA'et og dermed hjælper nukleosomsamlingsprocessen. Nogle histon chaperoner er specifikke for enten H2A / H2B eller H3 / H4, og nogle fungerer som chaperoner for begge. Denne protokol beskriver, hvordan in vitro-laboratorieteknikker såsom pull-down-assays, analytisk størrelsesekskluderingskromatografi, analytisk ultracentrifugering og histon chaperoning-assay kan bruges sammen for at bekræfte, om et givet protein er funktionelt som en histon chaperon.

Det rekombinante N-terminale nukleoplasmindomæne af proteinet FKBP53 fra Arabidopsis thaliana blev udsat for analytisk SEC. Elutionstopvolumenet blev plottet mod standardkurven for at identificere dets oligomere tilstand. De analytiske SEC-resultater afslørede, at domænet eksisterer som en pentamer i opløsning med en omtrentlig molekylmasse på 58 kDa (figur 1A, B). Endvidere blev nukleoplasmindomænet analyseret for termisk og kemisk stabilitet i forbindelse me...

Watch this Scientific Journal Video about In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays at JoVE.com

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Denne protokol beskriver et batteri af metoder, der inkluderer analytisk størrelsesekskluderingskromatografi til at studere histon chaperone oligomerisering og stabilitet, pull-down assay for at opklare histon chaperon-histon-interaktioner, AUC til at analysere støkiometrien af proteinkomplekserne og histon chaperoning assay for funktionelt at karakterisere en formodet histon chaperone in vitro.

In vitro Karakterisering af histon chaperoner ved hjælp af analytiske, pull-down og chaperoning assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Vores protokol beskriver, hvordan teknikker som pull-down assays analytisk størrelse ekskluderingskromatografi, analytisk ultracentrifugering og histon chaperoning assay kan bruges til at bekræfte, om et protein er en histon chaperon. De teknikker, der er dækket her, når de bruger tandem, kunne anvendes direkte til at karakterisere en formodet chaperone. hans kunne gå sammen med den strukturelle biologiske teknik.Teknikkerne kan anvendes inden for kromatinforskning. De enkelte metoder kan dog anvendes på ethvert andet protein af interesse efter behov. Surajit Gandhi, en ph.d.-studerende fra mit laboratorium, demonstrerer proceduren for den komplicerede pull-down assay;Archana Samal, en ph.d.-studerende, sammen med Mr. Ruchir C.Bobde for det analytiske ultracentrifugeringseksperiment;Mr. Somanath Baral, en ph.d.-studerende;sammen med Mr.Ketul Saharan for plasmidet super-coiling assay Bland først fem-mikromolære histon chaperoner med 20-mikromolære H2A / H2B dimers i 300 mikroliter ligevægtsbuffer. Histon chaperon H2A/H2B-komplekset centrifugeres for at fjerne eventuelt bundfald.Prøven indlæses på spinkolonnen, der er præ-ligestillet med ligevægtsbuffer. Kolonnen vaskes med 100 kolonnevolumener eller fire milliliter vaskebuffer for at fjerne overskydende H2A/H2B-dimer. Bland derefter histon chaperon H2A / H2B-komplekset med 20 til 60-mikromolære H3 / H4 tetramer.Kolonnen genvaskes med 100 kolonnevolumener eller fire milliliter vaskebuffer for at fjerne ubundet H3/H4-tetramer, og eluere derefter prøverne med elueringsbuffer. Udsæt de eluerede prøver for 18% SDS-SIDE. Plet derefter med Coomassie brilliant blue R250 og visualiser gelen.H2A/H2B-dimeren, H3/H4-tetrameren i histonchaperonen renses individuelt med dialysebufferen ved hjælp af analytisk størrelsesekskluderingskromatografi. Gem bufferen fra kørslen for at forberede yderligere fortyndinger senere og til brug som reference i AUC-cellen. For AUC-prøver blandes de oprensede chaperoner med dimer eller tetramer i separate rør til et endeligt volumen på 450 mikroliter ved hjælp af den gemte dialysebuffer for at nå en OD280 på 0,3 til 0,6.Saml cellen med et dobbeltsektor midterstykke og kvartsvinduer forsynet med en analytisk ultracentrifuge med en absorbansdetektor. Der fyldes 400 mikroliter af prøveopløsningen og 420 mikroliter dialysebuffer i henholdsvis reference- og prøvesektorerne i cellen. Vej og afbalancer cellerne nøjagtigt og læg dem i en fire-hullers titaniumrotor.Juster cellerne ved hjælp af mærkerne i bunden af cellerne og rotoren. Læg rotoren i centrifugen, luk låget, og start kørslen, hvilket gør det muligt for et vakuum at udvikle sig og temperaturen stabilisere sig. Til dataanalyse beregnes densitet og viskositet for proteinbufferkomponenterne og delvis specifikt volumen baseret på proteinets aminosyresammensætning ved hjælp af programmet SEDNTERP.Indlæs dataene fra AUC-maskinen i programmet SEDFIT. Definer menisken, den røde linje, cellebunden, blå linje og dataanalysegrænserne grønne linjer. Vælg Kontinuerlig CS-distribution som model.I parameterafsnittet skal du indstille opløsningsmaksimum op til 100. Indstil derefter sedimentationskoefficienten minimum til nul og maksimum til 10 til 15. Indstil friktionsforholdet til 1,2 oprindeligt, og vælg at flyde for at udlede forholdet fra dataene.Indstil F-forholdet til 0,68 for en sigma-regulering. Indstil delvis specifikt volumen, buffertæthed og viskositet som opnået fra SEDNTURP. Tryk på Kør for at tillade softwaren at løse lam-ligningen.Tryk på Tilpas for at finjustere. Vælg Vis peak Mw i CS i visningsfunktionen på hovedværktøjslinjen for at estimere molekylmasser. Bland to mikromolære H3/H4 tetramer og fire mikromolære H2A/H2B dimer med stigende koncentrationer af histon chaperon, der spænder fra en til seks mikromolar, i en samlingsbuffer til et slutvolumen på 50 mikroliter.Inkuber blandingen ved fire grader Celsius i 30 minutter. Samtidig forbehandles 500 nanogram af det negativt superoprullede PUC-19 plasmid med et mikrogram topoisomerase I-enzym i samlingsbufferen i et endeligt volumen ved 50 mikroliter. Kombiner derefter den forblandede opløsning indeholdende tetramer, dimer og histon chaperon med det afslappede plasma-DNA.Tilsæt 100 mikroliter 2X stopbuffer og inkuber for at afslutte samlingsreaktionen ved at deproteinisere plasmid-DNA'et. Tilsæt et lige stort volumen Tris-mættet phenol i røret indeholdende reaktionsblandingen og bland indholdet af reaktionsblandingen godt. Derefter centrifugeres prøven.Opsaml forsigtigt den øvre vandige fase indeholdende plasmid-DNA'et med en mikropipette og bland et lige stort volumen chloroform. Vortex blandingen. Derefter samles den øvre vandige fase, tilsæt et 1/10 volumen tre-molært natriumacetat med pH 5,5 og 2,5 volumener iskold ethanol.Bland opløsningen godt ved at vende røret tre til fire gange. Centrifugering af prøven ved 16.200 g i 10 minutter og kassér forsigtigt supernatanten. Hold rørene åbne ved stuetemperatur, indtil selv spormængder af ethanol fordamper, hvilket efterlader det udfældede plasmid-DNA i røret.De analytiske SEC-resultater afslørede, at det rekombinante N-terminale nukleoplasmindomæne af Arabidopsis thaliana-proteinet FKBP53 er en pentamer på 65 kilodalton i opløsning. Nukleoplasminprøven, der blev udsat for varmebehandling, viste, at domænet er meget termostabilt. Nukleoplasmindomænet viste saltstabilitet op til to molarer natriumchlorid og urinstofstabilitet op til fire molarer.Et pull-down-assay afslørede, at interaktionen mellem nukleoplasmindomænet og H2A/H2B-dimer var stabil op til 0,4 molær natriumchlorid, mens sammenhængen med H3/H4 var stabil op til 0,7 molarer. Chaperonen binder H2A/H2B dimer og H3/H4 tetramer samtidigt, uanset i hvilken rækkefølge de føjes til chaperonen, hvilket angiver separate interaktionssteder for oligomererne. De estimerede molekylmasser gennem AUC-SV afslører en 1: 1 støkiometri af nukleoplasmindomæne med histonoligomererne.I plasmid super-coiling assay øgede rekombinante plante histon chaperoner AtNRP1 og AtNRP2 mængden af super-oprullet plasmid, hvilket tyder på, at det kunne deponere histoner på DNA'et for at danne nukleosomer, hvilket forårsager DNA-superspolering. Plasmid super-coiling assay har brug for særlig pleje og mulig optimering for histon DNA og chaperone karakterisering. Isomer karakteriseringstelemetri kan bruges som opfølgning på analytiske størrelsesekskluderingseksperimenter for at bekræfte bindingsstabiliteten.

Research

JoVE Journal

Biochemistry

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

Species Determination and Quantitation in Mixtures Using MRM Mass Spectrometry of Peptides Applied to Meat Authentication

Artsbestemmelse og kvantificering i blandinger Brug MRM massespektrometri af peptider Anvendt til Kød Authentication

We describe a simple protocol for identifying and quantifying the two components in binary mixtures of species possessing one or more similar proteins. ...

Differential Scanning Calorimetry &#8212; A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differential Scanning Calorimetry ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Method for Assessing the Thermal Stability and Conformation of Protein Antigen

Differentialscanningskalometri - en metode til vurdering den termiske stabilitet og Kropsbygning af protein Antigen

Differential scanning calorimetry (DSC) is an analytical technique that measures the molar heat capacity of samples as a function of temperature. In the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Forbedret Prøve Multiplexing af væv under anvendelse kombinerede Precursor isotopmærkning og isobar Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Strukturel karakterisering af Mannan cellevæg polysaccharider i anlaeg, der anvender PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

Karakterisering af glykoproteiner med immunglobulin Fold af røntgenkrystallografi og biofysiske metoder

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

In vitro-allestedsnærværende og Deubiquitinationassays af Nukleosomale histoner

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Functional Characterization of RING-Type E3 Ubiquitin Ligases In Vitro and In Planta

Funktionel karakterisering af RING type E3 Ubiquitin-Ligaser in vitro og i planta

Ubiquitination, as a posttranslational modification of proteins, plays an important regulatory role in homeostasis of eukaryotic cells. The covalent ...

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Trinvis doseringprotokol for øget overførselstid i label-free impedansbaserede GPCR-analyser

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Karakterisering af Amyloid strukturer i Aging C. Elegans Brug Fluorescens Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin-specifikke tilpasninger af In vitro Fluorescens Mikroskopi-Baserede Motilitet Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...