Name: characterizing histone post-translational modification alterations in yeast neurodegenerative proteinopathy models
Uploaded: 2019-03-24T13:00:00
Description: Watch this Scientific Journal Video about Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models at JoVE.com

Vi takker Royena Tanaz, Huda Yousuf og Sadiqa Taasen for teknisk hjælp. Vi er meget taknemmelige for Prof. James Shorter den generøse bestemmelse af reagenser og intellektuelle bistand i udformningen af saccharose tuning eksperimenter. Gær plasmider var en generøs gave fra Prof. Aaron Gitler (herunder 303Gal-FUS; Addgene plasmid # 29614). Brooklyn College samt den avancerede videnskab forskning Center (CUNY) og en NIH NINDS avanceret postdoc overgang Award (K22NS09131401) understøttes M.P.T.

1. omdanne S. cerevisiae med neurodegenerative proteinopathy-forbundet protein konstruktioner
<ol><li>Vokse vildtype (WT) 303 gær i gær extract pepton dextrose (YPD) bouillon natten under omrystning (200 rpm) ved 30 ° C.</li>
	<li>Efter 12−16 h af vækst, fortyndes gær til en optisk tæthed på 600 nm (OD600) på 0,25 med YPD. 10 mL af gær flydende kultur vil være nødvendigt for hver transformation, forberede fem transformationer svarende til FUS, TDP-43, en synuclein, og vektor kun (ccdB) ...

<ol>
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Protokollen beskrevet her giver en enkel, hensigtsmæssig og omkostningseffektiv måde at kategorisere genome-wide Histon PTM ændringer korreleret med neurodegenerative proteinopathies. Mens der er andre modeller af ALS og PD, såsom in vitro- modeller humane cellelinjer og murine32, S. cerevisiae forbliver attraktivt på grund af sin brugervenlighed. For eksempel gær modeller kræver ikke brug af en steril hætte, eller har de brug for den intensive træning, går sammen med c...

Neurodegenerative sygdomme er ødelæggende sygdomme med lidt at ingen behandlingsmuligheder til rådighed. Blandt disse er Amyotrofisk lateral sklerose (ALS) og Parkinsons sygdom (PD) særligt forfærdelige. Ca. 90% af ALS og PD tilfælde betragtes som sporadiske, forekommende uden familie historie af sygdommen, mens de resterende sager køre i familier og er normalt knyttet til et specifikt gen mutation1,2. Interessant, er begge af disse sygdomme forbundet med protein mislocalization og sammenlægning3,4,5,6. Eksempelvis, sammenvokset i sarkom (FUS) og TAR DNA-bindende protein 43 (TDP-43) er RNA-bindende proteiner, der mislocalize til cytoplasma og samlet i ALS7,8,9,10, 11,12, mens α-synuclein er princippet komponent af proteinholdige aggregater betegnes Lewy organer i PD5,13,14,15.
Trods den omfattende genome-wide association indsats i store patientgrupper forblive det overvældende flertal af ALS og PD tilfælde uforklarlige genetisk. Epigenetik kan spille en rolle i neurodegenerativ sygdom? Epigenetik omfatter ændringer i genekspression forekommende uden ændringer til underliggende DNA sekvens16. En vigtigste epigenetiske mekanisme indebærer de indlæg translationel ændringer (PTMs) af Histon proteiner16. I eukaryote celler og er genetisk materiale tæt pakket ind i kromatin. Basisenhed af kromatin er nucleosome, bestående af 146 basepar af DNA snoet omkring en Histon octamer, sammensat af fire par histoner (to eksemplarer hver histoner H2A, H2B, H3 og H4)17. Hver Histon har en N-terminale hale, der stikker ud af nucleosome og kan blive ændret ved tilsætning af forskellige kemiske grupper, som regel på Lysin og arginin rester18. Disse PTMs er dynamisk, hvilket betyder, at de kan nemt tilføjes og fjernes, og omfatter grupper såsom acetylation, methylering og fosforylering. PTMs kontrollere tilgængelighed af DNA på transkriptionel maskinen, og dermed hjælpe kontrol gen expression18. For eksempel Histon acetylation reducerer styrken af den elektrostatiske interaktion mellem de meget grundlæggende histoneprotein og negativt ladede DNA rygraden, så generne pakket af acetyleret histonerne være mere tilgængelige og dermed meget udtrykt19. For nylig, den bemærkelsesværdige biologiske specificiteten af særlig Histon PTMs og kombinationer heraf har ført til den Histon kode hypotese20,21 i hvilke proteiner, skrive, slette, og læse Histon PTMs alle handle i fællesskab for at modulere genekspression.
Gær er en meget nyttig model til at studere neurodegeneration. Vigtigere, bevares mange neuronal cellulære veje fra gær til mennesker22,23,24. Gær sammenfatte cytotoksicitet fænotyper og protein inklusioner ved overekspression af FUS, TDP-43 eller α-synuclein22,23,24,25,26. I virkeligheden, har Saccharomyces cerevisiae modeller af ALS været brugt til at identificere genetiske risikofaktorer i mennesker27. Derudover gær overekspression menneskelige α-synuclein tilladt til karakterisering af Rsp5 netværk som druggable mål at forbedre α-synuclein toksicitet i neuroner28,29.
Her, beskriver vi en protokol at udnytte Saccharomyces cerevisiae ægprodukters genome-wide Histon PTM ændringer forbundet med neurodegenerative proteinopathies (figur 1). Brugen af S. cerevisiae er særdeles attraktivt på grund af sin brugervenlighed, lav pris, og hastighed i forhold til andre in vitro og animalske modeller af neurodegeneration. Udnyttelse af tidligere udviklet ALS og PD modeller22,23,25,26, vi har overexpressed menneskelige FUS, TDP-43 og α-synuclein i gær og udækkede særskilte Histon PTM forandringer i forbindelse med hver proteinopathy30. Den protokol, som vi beskriver her kan være afsluttet i mindre end to uger fra transformation til dataanalyse.

<table>
	<tbody>
		<tr>
			<td>-His DO Supplement</td>
			<td>Clontech</td>
			<td>630415</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Running Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 141.65 g glycine (ThermoFisher BP381-1), 30.3 g Tizma base (Sigma-Aldrich T6066), 10 g sodium dodecyl sulfate (Sigma-Aldrich L3771), and 1 L deionized water, pH 8.8.</td>
		</tr>
		<tr>
			<td>12% Polyacrylamide Gels</td>
			<td>BIO-RAD</td>
			<td>456-1041</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H3 (Lys14) Primary Antibody</td>
			<td>MilliporeSigma</td>
			<td>07-353 (Lot No. 2762291)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys 16) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab109463 (Lot No. GR187780)</td>
			<td>Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys12) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab46983 (Lot No. GR71882)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Anti-dimethyl-Histone H3 (Lys36) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab9049 (Lot No. GR266894, GR3236147)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-Histone H3 Primary Antibody</td>
			<td>Abcam</td>
			<td>ab24834 (Lot No. GR236539, GR174196, GR3194335)</td>
			<td>Nuclear Loading Control; Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2B (Thr129) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab188292 (Lot No. GR211874)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3 (Ser10) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab5176 (Lot No. GR264582, GR192662, GR3217296)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>BioPhotometer D30</td>
			<td>Eppendorf</td>
			<td>6133000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish (100 x 20 mm)</td>
			<td>Eppendorf</td>
			<td>30702118</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate, 96 well</td>
			<td>Eppendorf</td>
			<td>30730011</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804/5804 R/5810/5810 R</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IRDye 800 CW</td>
			<td>LI-COR</td>
			<td>926-32212 (Lot No. C60301-05, C61116-02, C80108-05)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IRDye 860 RD</td>
			<td>LI-COR</td>
			<td>926-68073 (Lot No. C60217-06, C70323-06, C70601-05, C80116-07)</td>
			<td>Dilution: 1/2500</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra thick blot paper (filter paper)</td>
			<td>BIO-RAD</td>
			<td>1703968</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Prepare 50 % w/v solution.</td>
		</tr>
		<tr>
			<td>Immobilon-FL Transfer Membranes</td>
			<td>MilliporeSigma</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium acetate dihydrate (LiAc)</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td>Prepare a 1 M solution.</td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td></td>
			<td></td>
			<td>Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue (Sigma-Aldrich B8026), 4.7 mL glycerol, 1.2 mL 0.5M Trizma base pH 6.8, 0.93 g DL-Dithiothreitol (Sigma-Aldrich D0632), and 2.1 mL deionized water.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoeresis Cell</td>
			<td>BIO-RAD</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>2231300045</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Restriction Enzyme Buffer 2.1, 10x</td>
			<td>New England Bio Labs</td>
			<td>102855-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Nhe I Restriction Enzyme</td>
			<td>New England Bio Labs</td>
			<td>101228-710</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free Water</td>
			<td>Qiagen</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc Imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>2800-03</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniTray Cell Culture Treated w/Lid Sterile, PS (86 x 128 mm)</td>
			<td>ThermoFisher</td>
			<td>165218</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-a-synuclein-GFP</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-ccdB</td>
			<td>Addgene</td>
			<td>14133</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303Gal-FUS</td>
			<td>Addgene</td>
			<td>29614</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-TDP-43</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) (PEG)</td>
			<td>Sigma-Aldrich</td>
			<td>P4338</td>
			<td>Prepare a 50% w/v solution.</td>
		</tr>
		<tr>
			<td>Ponceau S Stain</td>
			<td>Sigma-Aldrich</td>
			<td>P3504</td>
			<td>Mix: 0.5 g 0.1% w/w Ponceau S dye, 5 mL 1% v/v acetic acid (Sigma-Aldrich 320099), and 500 mL deionized water.</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BIO-RAD</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>R7630</td>
			<td>Prepare 10% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Agilent Tech</td>
			<td>201190</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar (Sigma-Aldrich A1296), 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids (ThermoFisher 291920), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>SGal-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar, 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids, 100 mL galactose solution, and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate Loading Buffer</td>
			<td></td>
			<td></td>
			<td>Store at -20 oC. 6X, Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue, 0.93 g DL-Dithiothreitol, 2.1 mL deionized water, 4.7 mL glycerol, and 1.2 mL 0.5 M Trizma base, pH 6.8.</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>Prepare 0.2 M solution.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>TBS + 0.1% Tween 20 (TBST)</td>
			<td></td>
			<td></td>
			<td>Mix: 100 mL 10X TBS, 1 mL Tween 20 (Sigma-Aldrich P7949), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>TBS Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td>BIO-RAD</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 22.5 g glycine, 4.84 g Tizma base, 400 mL methanol, 1 g sodium dodecyl sulfate, and 1.6 L deionized water.</td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline (TBS)</td>
			<td></td>
			<td></td>
			<td>10X, 7.6 pH, Solution: Mix 24 g Trizma base, and 88 g sodium chloride (Sigma-Aldrich S7653). Fill to 1 L with deionized water.</td>
		</tr>
		<tr>
			<td>WT 303 S. cerevisiae yeast</td>
			<td>Gift from J. Shorter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose (YPD)</td>
			<td>Sigma-Aldrich</td>
			<td>Y1375</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing histone post-translational modification alterations in yeast neurodegenerative proteinopathy models

Neurodegenerative sygdomme som Amyotrofisk lateral sklerose (ALS) og Parkinsons sygdom (PD), forårsage tab af hundreder af tusinder af liv hvert år. Der mangler effektive behandlingsmuligheder at standse sygdomsprogression. Trods den omfattende sekventering indsats i store patientgrupper forblive størstedelen af ALS og PD tilfælde uforklarlige af genetiske mutationer alene. Epigenetik mekanismer, såsom den posttranslationel modifikation af Histon proteiner, kan være involveret i neurodegenerativ sygdom ætiologi og progression og føre til nye mål for farmaceutisk intervention. Pattedyr in vivo og in vitro-modeller af ALS og PD er dyre og kræver ofte langvarig og besværlig eksperimentelle protokoller. Her, skitsere vi en praktiske, hurtig og omkostningseffektiv fremgangsmåde til bestemmelse af genome-wide ændringer i Histon ændring niveauer ved hjælp af Saccharomyces cerevisiae som et modelsystem. Denne protokol giver mulighed for omfattende undersøgelser af epigenetiske ændringer tilsluttet neurodegenerative proteinopathies, der underbygger tidligere resultater i forskellige modelsystemer mens betydeligt udvide vores kendskab til den neurodegenerativ sygdom epigenome.

For at illustrere denne metode, vil vi drage fordel af Seneste offentliggjorte resultater30. WT menneskelige FUS og TDP-43 var overexpressed for 5 h, mens WT α-synuclein var overexpressed for 8 h. En ccdB konstruktion blev brugt som en vektor negativ kontrol. Figur 2 viser vækst undertrykkelse i faste og flydende kulturer. Gær blev høstet som beskrevet og Western blotting med modifikation-specifikke antistoffer blev udført. Anti-t...

Watch this Scientific Journal Video about Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models at JoVE.com

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Denne protokol beskriver eksperimentelle procedurer for at karakterisere genome-wide ændringer i niveauet af Histon posttranslationelle modifikationer (PTM) forekommer i forbindelse med overekspression af proteiner er forbundet med ALS og Parkinsons sygdom i Saccharomyces cerevisiae modeller. Efter SDS-PAGE adskillelse registreres individuelle Histon PTM niveauer med modifikation-specifikke antistoffer via Western blotting.

Kendetegner Histon posttranslationel modifikation ændringer i gær Neurodegenerative Proteinopathy modeller

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Denne protokol giver os mulighed for nemt at udforske de epigenetiske træk ved neurodegenerative sygdomme. Den største fordel ved denne teknik er, at det udnytter gær som en model system, i forhold til andre cellulære og animalske modeller af neurodegenerative sygdom, gær er hensigtsmæssigt og omkostningseffektiv. Denne teknik giver os mulighed for at kortlægge de epigenetiske ændringer, der opstår i neurodegenerative sygdomme, hvilket kan føre til nye markører for diagnose, samt nye mål for terapi.Denne metode gør det muligt yderligere at undersøge de epigenetiske mærkers rolle i neurodegenerative sygdomme og kan ændres til vurdering af humane fibroblaster og inducerede pluripotente stamceller. Denne særlige metode indeholder en række detaljer, der skal demonstreres visuelt, såsom korrekt lastning af western blot apparat. Demonstrere proceduren vil være Michel Fallah og Navin Rana, begge bachelor forskere i Torrente laboratorium.Begynd med at restreaking gær fra frosne glycerol bestande på histidin 2% glukose agar selektiv medium plader, for en to til tre dages inkubation ved 30 grader Celsius. Ved inkubationens afslutning skal der vaccineres restreakeret gær for hver overpressionsmodel og kontrolleres i fem milliliter histidinseltivt flydende medium. Suppleret med 2% raffinose ved 30 grader Celsius og 200 rotationer i minuttet, natten over.Den næste morgen, vokse 100 milliliter flydende kultur for hver af de overekspression modeller og kontrol i selektive medier suppleret med 2% galactose natten over. Og måle den optiske tæthed ved 600 nanometer eller OD600 for at bestemme mængden af natten kultur er nødvendig for at gøre over udtryk kulturer på en start OD600 af 0,3. For at fremkalde protein overekspression, dyrke gær på galactose medium med omrystning ved 30 grader Celsius for den relevante eksperimentelle periode.Mål derefter OD600 for hver kultur ved slutningen af induktionsperioden. Og standardisere alle celletal til den laveste OD600-værdi. Høst kulturerne i 50 milliliter centrifugerør ved centrifugering og resuspenderede pellets i en milliliter sterilt stillantvand for hver 10 milliliter dyrket kultur.Aliquot en milliliter celle resuspension i individuelle mikrocentrifuge rør for en ekstra centrifugering og aspirere supernatanter. Derefter fastfryse cellepillerne i flydende nitrogen til opbevaring ved minus 80 grader celsius. At lyse cellerne til Western blot analyse, tø gærcelle pellets på is før resuspension i 100 mikroliter af destilleret vand.Der tilsættes 300 mikroliter 0,2-molar natriumhydroxid og 20 mikroliter 2-Mercaptoethanol til hver celleprøve. Og resuspend pellets ved pipetting. Efter 10 minutters inkubation på is pellets cellerne ved centrifugering og resuspenderede prøverne i 100 mikroliter af lastefarve.Derefter koges prøverne i 10 minutter på en varmeblok. Mens prøverne lyses, skal du placere to geler i en gelholder og fylde det indre kammer til toppen med løbebuffer og yderkammeret til togellinjemærket. Når prøverne er klar, belastning 15 mikroliter hver, celle lysis opløsning, i hver brønd af de 10 godt, 12% polyacrylamid gel og tilsæt fem mikroliter af protein stigen til protein stigen lane godt.Kør gelen i ca 45 minutter ved 150 volt eller indtil lastning farvestof front når bunden af gelen. Mens gelen kører, sættetid to fiber puder pr gel i overførsel buffer og en polyvinyliden fluorid, eller PVDF, membran pr gel i methanol. Når gelen er klar, skylles membranen i overførselsbufferen, og en presoaked fiberpude anbringes i bunden af en halvtørre overførselsapparatcelle.Efterfulgt af PVDF membranen, gelen og den anden presoaked fiber pad. Derefter indstilles strømmen til 150 milliampere i en time. Ved afslutningen af proteinoverførslen fjernes membranen fra overførselsapparatet for at få en kort skylning med destilleret vand.Placer den skyllede membran, protein side op i en lille farvning boks og dække membranen med tris-buffered saltvand eller TBS, blokering buffer for en time inkubation ved stuetemperatur med blid rokkende. Ved afslutningen af blokering inkubationen, inkubere membranen natten over med en anti-gær histon og modifikation-specifikke antistof i TBS ved fire grader Celsius. Og en ordentlig nuklear lastning kontrol antistof.Den næste morgen, vaske membranen fire gange i TBS, suppleret med 0,1% polysorbat 20 i fem minutter med rokkende ved stuetemperatur per vask. Efter den sidste vask inkuberes skamplet med de relevante sekundære antistoffer i en time ved stuetemperatur. Efterfulgt af fire, fem minutter vasker i TBST og en fem minutters vask i TBS alene med vuggende.Derefter billede skamplet på en fluorescerende vestlige blot forestille system i to minutter. Her, vækst undertrykkelse i faste og flydende kulturer er vist med betydelige virkninger på gær vækst observeret i tilstedeværelsen af galactose-infunderes i sarkom overexpression modeller. Et betydeligt fald i histonniveauer er synlige i den sammensmeltede i sarkomoverekpressionsmodellen og signifikante stigninger i niveauet af histonacetylation i TAR-bindingsproteinet 43 overekspressionsmodel, som ikke ses i hverken den sammensmeltede i sarkom eller alpha synuclein overekspressionsmodellen.Der er også betydelige fald i histone niveauer i alfa synuclein overexpression model. I dette saccharosetument, jo lavere er mængden af galactose, der anvendes til at fremkalde sammensmeltet i sarkom overekspression, den lavere toksicitet, der observeres i både fast og flydende kultur. Vigtigere, jo lavere niveau af smeltet i sarkom overexpression, jo mindre er størrelsen af reduktionen i histone niveauer.Mens du udfører denne protokol, er det bydende nødvendigt at standardisere mængden af gær i hver prøve for at muliggøre en korrekt sammenligning i niveauet af histone post-translationel ændring. 2-Mercaptoethanol bør anvendes under en hætte for at undgå indånding. Hvis der anvendes Ponceau-pletten, skal den kasseres korrekt.

Research

JoVE Journal

Genetics

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Optogenetic Tilfældig mutagenese ved anvendelse histon-miniSOG i<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Påvisning af Copy Number Ændringer Brug Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Målrettet<em&gt; I Situ</em&gt; Mutagenese af histon-gener i knopskydende gær

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Konstituerende og inducerbar systemer for genetiske In Vivo ændring af mus hepatocytter brug af hydrodynamiske hale vene injektion

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Kromatin Immunoprecipitation (ChIP) af Histon ændringer fra Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Gen-målrettet tilfældig mutagenese at vælge Heterochromatin-destabiliserende proteasom mutanter i Fission gær

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

En Fast og kvantitativ metode til posttranslationel modifikation og Variant aktiveret kortlægning af peptider til genomer

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

Enkelt molekyle fluorescens In Situ hybridisering (smFISH) analyser i spirende gær vegetativ vækst og meiose

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Mating-based Overexpression Library Screening in Yeast

Parring-baserede overekspression bibliotek Screening i gær

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Forberedelse og gen modifikation af ikkemenneske primat hæmatopoietisk stamceller og stamceller

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...