Name: rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag
Uploaded: 2017-12-14T14:00:00
Description: Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

Vi takker Dr. Takeshi Iwatsubo (University of Tokyo, Japan) venligst give plasmider kodning af 3xFLAG-LRRK2 WT og mutanter. Vi takker også Dr. Dario Alessi (universitetet i Dundee, UK) venligst give MLi-2 og plasmid kodning HA-Rab10. Dette arbejde blev støttet af Japan-samfund til fremme af videnskab (JSP'ER) KAKENHI tilskud antal JP17K08265 (G.I.).

1. Prøvetilberedning for P-tag SDS-PAGE
<ol>
	<li>Fjern og kassér medier fra 10 cm retter hvor celler dyrkes ved hjælp af en suge og vaske cellerne med 5 mL Dulbecco fosfatbufferet saltopløsning (DPBS) af første tilføjer DPBS til siden af retter at undgå at forstyrre cellelag og manuelt rock retter tilbage og tilbage flere gange.</li>
	<li>Fjerne og kassere DPBS ved hjælp af en suge og der tilsættes 2 mL 0,25% (w/v) trypsin fortyndet i DPBS, og forsigtigt rock retter for at dække cellelag. Sætte retterne i e...

<ol>
	<li>Sveinbjornsdottir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+symptoms+of+Parkinson's+disease.">The clinical symptoms of Parkinson's disease.</a> J. Neurochem. 139 (Suppl. 1), 318-324 (2016).</li><li>Hernandez, D. G., Reed, X., Singleton, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+in+Parkinson+disease:+Mendelian+versus+non-Mendelian+inheritance.">Genetics in Parkinson disease: Mendelian versus non-Mendelian inheritance.</a> J. Neurochem. 139 (Suppl. 1), 59-74 (2016).</li><li>Pais&#225;n-Ru&#237;z, C., 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=Cloning+of+the+gene+containing+mutations+that+cause+PARK8-linked+Parkinson's+disease.">Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease.</a> Neuron. 44 (4), 595-600 (2004).</li><li>Zimprich, 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=Mutations+in+LRRK2+cause+autosomal-dominant+parkinsonism+with+pleomorphic+pathology.">Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology.</a> Neuron. 44 (4), 601-607 (2004).</li><li>Gilks, W. 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=A+common+LRRK2+mutation+in+idiopathic+Parkinson's+disease.">A common LRRK2 mutation in idiopathic Parkinson's disease.</a> Lancet. 365 (9457), 415-416 (2005).</li><li>Satake, W., 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=Genome-wide+association+study+identifies+common+variants+at+four+loci+as+genetic+risk+factors+for+Parkinson's+disease.">Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease.</a> Nat. Genet. 41 (12), 1303-1307 (2009).</li><li>Sim&#243;n-S&#225;nchez, J., 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=Genome-wide+association+study+reveals+genetic+risk+underlying+Parkinson's+disease.">Genome-wide association study reveals genetic risk underlying Parkinson's disease.</a> Nat. Genet. 41 (12), 1308-1312 (2009).</li><li>Klein, C., Ziegler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imputation+of+sequence+variants+for+identification+of+genetic+risks+for+Parkinson's+disease:+a+meta-analysis+of+genome-wide+association+studies.">Imputation of sequence variants for identification of genetic risks for Parkinson's disease: a meta-analysis of genome-wide association studies.</a> Lancet. 377 (9766), 641-649 (2011).</li><li>Cookson, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+leucine-rich+repeat+kinase+2+(LRRK2)+in+Parkinson's+disease.">The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson's disease.</a> Nat. Rev. Neurosci. 11 (12), 791-797 (2010).</li><li>Ozelius, L. J., 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=LRRK2+G2019S+as+a+Cause+of+Parkinson’s+Disease+in+Ashkenazi+Jews.">LRRK2 G2019S as a Cause of Parkinson’s Disease in Ashkenazi Jews.</a> N. Engl. J. Med. 354 (4), 424-425 (2006).</li><li>Lesage, 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=LRRK2+G2019S+as+a+Cause+of+Parkinson’s+Disease+in+North+African+Arabs.">LRRK2 G2019S as a Cause of Parkinson’s Disease in North African Arabs.</a> N. Engl. J. Med. 354 (4), 422-423 (2006).</li><li>Bouhouche, 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=LRRK2+G2019S+Mutation:+Prevalence+and+Clinical+Features+in+Moroccans+with+Parkinson's+Disease.">LRRK2 G2019S Mutation: Prevalence and Clinical Features in Moroccans with Parkinson's Disease.</a> Parkinsons. Dis. , 1-7 (2017).</li><li>West, A. B., 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=Parkinson's+disease-associated+mutations+in+leucine-rich+repeat+kinase+2+augment+kinase+activity.">Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (46), 16842-16847 (2005).</li><li>Ito, G., 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=GTP+binding+is+essential+to+the+protein+kinase+activity+of+LRRK2,+a+causative+gene+product+for+familial+Parkinson's+disease.">GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease.</a> Biochemistry. 46 (5), 1380-1388 (2007).</li><li>Steger, 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=Phosphoproteomics+reveals+that+Parkinson's+disease+kinase+LRRK2+regulates+a+subset+of+Rab+GTPases.">Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.</a> Elife. 5, (2016).</li><li>Ito, G., 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=Phos-tag+analysis+of+Rab10+phosphorylation+by+LRRK2:+a+powerful+assay+for+assessing+kinase+function+and+inhibitors.">Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.</a> Biochem. J. 473, 2671-2685 (2016).</li><li>Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K., Koike, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-binding+tag,+a+new+tool+to+visualize+phosphorylated+proteins.">Phosphate-binding tag, a new tool to visualize phosphorylated proteins.</a> Mol. Cell. Proteomics. 5 (4), 749-757 (2006).</li><li>Using a Hemacytometer to Count Cells. J. Vis. Exp Available from: <a target="_blank" href="https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells">https://www.jove.com/science-education/5048/using-a-hemacytometer-to-count-cells</a> (2017)</li><li>Ni, D., Xu, P., Gallagher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+Immunodetection.">Immunoblotting and Immunodetection.</a> Curr. Protoc. Mol. Biol. (114), 10.8.1-10.8.37 (2016).</li><li>Reith, A. 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=GSK2578215A;+a+potent+and+highly+selective+2-arylmethyloxy-5-substitutent-N-arylbenzamide+LRRK2+kinase+inhibitor.">GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.</a> Bioorg. Med. Chem. Lett. 22 (17), 5625-5629 (2012).</li><li>Fell, M. J., 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=MLi-2,+a+potent,+selective+and+centrally+active+compound+for+exploring+the+therapeutic+potential+and+safety+of+LRRK2+kinase+inhibition.">MLi-2, a potent, selective and centrally active compound for exploring the therapeutic potential and safety of LRRK2 kinase inhibition.</a> J. Pharmacol. Exp. Ther. 355, 397-409 (2015).</li><li>Dzamko, N., 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=Inhibition+of+LRRK2+kinase+activity+leads+to+dephosphorylation+of+Ser(910)/Ser(935),+disruption+of+14-3-3+binding+and+altered+cytoplasmic+localization.">Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.</a> Biochem. J. 430 (3), 405-413 (2010).</li><li>Th&#233;venet, J., Pescini Gobert, R., Hooft van Huijsduijnen , R., Wiessner, C., Sagot, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+LRRK2+expression+points+to+a+functional+role+in+human+monocyte+maturation.">Regulation of LRRK2 expression points to a functional role in human monocyte maturation.</a> PLoS One. 6 (6), e21519 (2011).</li></ol>

Her, beskriver vi en letkøbt og robust metode til påvisning af Rab10 fosforylering af LRRK2 på endogene niveauer baseret på metoden P-tag. Fordi den foreliggende antistof mod fosforyleres Rab10 virker kun med overexpressed proteiner15, er den nuværende metode udnytter P-tag SDS-PAGE den eneste måde at vurdere endogene niveauer af Rab10 fosforylering. Desuden, den nuværende metode giver mulighed for vurdering af støkiometrisk af Rab10 fosforylering i celler. Fordi metoden P-tag er generisk ...

PD er et af de mest almindelige neurodegenerative sygdomme, overvejende påvirker dopaminerge neuroner i midthjernen, der resulterer i dysfunktion af motoriske systemer i ældre1. Mens de fleste patienter udvikler PD i en sporadisk måde, er der familier arve sygdommen. Mutationer i flere gener er blevet fundet at være forbundet med FPD2. En af de sygdomsfremkaldende gener for FPD er LRRK2, hvor otte missense mutationer (N1437H, R1441C/G/H/S, Y1699C, G2019S og I2020T) knyttet til en overvejende nedarvede FPD kaldet PARK8 har hidtil været rapporteret3,4,5. Flere genome-wide association studier (GWAS) af sporadiske PD patienter har også identificeret genomisk variationer i LRRK2 locus som en risikofaktor for PD, tyder på, at abnormitet i funktionen af LRRK2 er en almindelig årsag til neurodegeneration i både sporadisk og PARK8 FPD6,7,8.
LRRK2 er et stort protein (2,527 aminosyrer) bestående af en leucin-rig gentage domæne, et GTP-bindende Ras af komplekse proteiner (ROC) domæne, en C-terminale ROC (COR) domæne, en Serin/Threonin protein kinase domæne og en WD40 gentage domæne9. De otte FPD mutationer finde i disse funktionelle domæner; N1437H og R1441C/G/H/S i domænet ROC, Y1699C i domænet ru, G2019S og I2020T i domænet kinase. Da G2019S mutationen, som er det hyppigst fundet mutation i PD patienter10,11,12, øger kinase aktivitet af LRRK2 af 2-3 fold in vitro-13, det er en hypotese at den unormale stigning i fosforylering af LRRK2 substrate(s) er giftigt for neuroner. Dog har det været umuligt at studere om fosforylering af fysiologisk relevante LRRK2 substrater er ændret i familiær/sporadisk PD patienter på grund af manglende metoder evaluerer det i afledte patientprøver.
Protein fosforylering er generelt fundet ved immunoblotting eller enzymmaerket assay (ELISA) ved hjælp af antistoffer specielt anerkende fosforyleres tilstand af proteiner eller af massespektrometriske analyse. Men den tidligere strategi sommetider ikke kan anvendes på grund af vanskeligheder med at skabe fosforylering-specifikke antistoffer. Metaboliske mærkning af celler med radioaktivt fosfat er en anden mulighed at undersøge fysiologiske niveauer af fosforylering når fosforylering-specifikke antistoffer ikke er let tilgængelige. Men det kræver en stor mængde af radioaktivt materiale og derfor involverer nogle specialiserede udstyr for strålingsbeskyttelse14. Massespektrometrisk analyse er mere følsomme i forhold til disse immunokemisk metoder og blev populær i at analysere protein fosforylering. Dog prøveforberedelsen er tidskrævende og dyre instrumenter er påkrævet til analysen.
Et undersæt af Rab GTPase familie, herunder Rab10 og Rab8 blev for nylig rapporteret som direkte fysiologiske substrater for LRRK2 baseret på resultatet af en storstilet phosphoproteomic analyse15. Vi viste så, at Rab10 fosforylering blev øget ved FPD mutationer i mus embryonale fibroblaster og i lungerne af knockin mus16. I denne rapport, valgte vi at ansætte en sodium dodecyl sulfat polyacrylamid gelelektroforese (SDS-PAGE)-baseret metode, hvor en P-tag molekyle er Co polymere i SDS-PAGE geler (P-tag SDS-PAGE) til påvisning af de endogene niveauer af Rab10 fosforylering, fordi en yderst følsom antistoffer specifikke for fosforyleres Rab10 manglede stadig. Vi har undladt at registrere fosforylering af endogene Rab8 på grund af dårlig selektivitet i øjeblikket tilgængelige antistoffer for total Rab8. Derfor besluttede vi at fokusere på Rab10 fosforylering. LRRK2 phosphorylates Rab10 på Thr73 at finde i midten af regionen meget bevaret "skifte II". Høj bevarelse af fosforylering sites blandt Rab proteiner kan være en af grundene til hvorfor phosphospecific antistoffer anerkende særskilte Rab proteiner er vanskelige at gøre.
Fosforylering af Rab8A af LRRK2 hæmmer bindingen af Rabin8, en guanin nukleotid udveksling faktor (GEF) som aktiverer Rab8A ved at udveksle de bundne BNP med GTP15. Fosforylering af Rab10 og Rab8A af LRRK2 hæmmer også binding af BNP-dissociation hæmmere (GDIs), som er nødvendige for aktivering af Rab proteiner ved at udtrække BNP-bundet Rab proteiner fra membraner15. Kollektivt, er det en hypotese at fosforylering af Rab proteiner af LRRK2 forhindrer dem i aktivering, selv om de præcise molekylære mekanisme og fysiologiske konsekvenser af fosforylering forbliver uklart.
P-tag SDS-PAGE blev opfundet af Kinoshita et al. i 2006: I denne metode, acrylamid var kovalent kombineret med P-tag, et molekyle, indfange fosfater med høj affinitet, som copolymeriseret i SDS-PAGE geler17. Fordi P-tag molekyler i en SDS-PAGE gel retard selektivt elektroforese mobilitet af fosforyleres proteiner, P-tag SDS-PAGE kan adskille fosforyleres proteiner fra ikke-fosforyleret dem (figur 1). Hvis protein af interesse er fosforyleret på flere restprodukter, vil en stige af bands svarende til varierende fosforyleres formularer observeres. I forbindelse med Rab10 observere vi kun én skiftede bandet, der angiver, at Rab10 er fosforyleret kun på Thr73. Den største fordel ved P-tag SDS-PAGE over immunoblotting med fosforylering-specifikke antistoffer er at fosforyleres Rab10 kan påvises ved immunoblotting med ikke-fosforylering-specifikke antistoffer (dvs., erkender samlede Rab10) efter at være overført på membraner, er der generelt mere specifikke, følsomme og tilgængelig fra kommercielle/akademiske kilder. En anden fordel ved at bruge P-tag SDS-PAGE er at man kan få omtrentlige estimering af støkiometrisk af fosforylering, hvilket er umuligt ved immunoblotting med fosforylering-specifikke antistoffer eller metaboliske mærkning af celler med radioaktivt fosfater.
Ud over brugen af billig P-tag acrylamid og nogle mindre ændringer relateret til det, følger den nuværende metode til påvisning af Rab10 fosforylering af LRRK2 en generel protokol af immunoblotting.Derfor bør det være ligetil og nemt eksekverbare i alle laboratorier, hvor immunoblotting er en sædvanlig praksis, med alle typer af prøver herunder renset proteiner, cellelysater og væv homogeniseret.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>homemade</td>
			<td></td>
			<td>150 mM NaCl, 8 mM Na2HPO4-12H2O, 2.7 mM KCl, 1.5 mM KH2PO4 in MilliQ water and sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Nacalai Tesque</td>
			<td>31320-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Disodium Hydrogenphosphate 12-Water</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Wako</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>Wako</td>
			<td>169-04245</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Trypsin (10X)</td>
			<td>Sigma-Aldrich</td>
			<td>T4549</td>
			<td>Dilute 10-fold with sterile DPBS for preparing working solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle medium 
			(DMEM)</td>
			<td>Wako</td>
			<td>044-29765</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>BioWest</td>
			<td>S1560</td>
			<td>Heat-inactivated at 56 &#176;C for 30 min</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Wako</td>
			<td>342-01375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Wako</td>
			<td>198-13765</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine HCl MAX, Linear, Mw 40,000 (PEI MAX 40000)</td>
			<td>PolySciences, Inc.</td>
			<td>24765-1</td>
			<td>Stock solution was prepared in 20 mM HEPES-NaOH pH 7.0 at 1 mg/mL and the pH was then adjusted to 7.0 with NaOH</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Wako</td>
			<td>045-28335</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>STAR</td>
			<td>RSP-THA500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Wako</td>
			<td>080-01066</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene(10) Octylphenyl Ether</td>
			<td>Wako</td>
			<td>160-24751</td>
			<td>Equivalent to Triton X-100</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8217;,N&#8217;-tetraacetic acid (EGTA)</td>
			<td>Wako</td>
			<td>346-01312</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium orthovanadate(V)</td>
			<td>Wako</td>
			<td>198-09752</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>Kanto Chemical</td>
			<td>37174-20</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Glycerophosphoric Acid Disodium Salt Pentahydrate</td>
			<td>Nacalai Tesque</td>
			<td>17103-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate decahydrate</td>
			<td>Kokusan Chemical</td>
			<td>2113899</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcystin-LR</td>
			<td>Wako</td>
			<td>136-12241</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Dissolve one tablet in 1 mL water, which can be stored at -20 &#176;C for a month. Use it at 1:50 dilution for cell lysis</td>
		</tr>
		<tr>
			<td>Pierce Coomassie (Bradford) Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Nacalai Tesque</td>
			<td>31607-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Wako</td>
			<td>075-00616</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Wako</td>
			<td>021-02911</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Kanto Chemical</td>
			<td>25099-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>056-06967</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako</td>
			<td>136-01837</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-binding tag acrylamide</td>
			<td>Wako</td>
			<td>AAL-107</td>
			<td>P-tag acrylamide</td>
		</tr>
		<tr>
			<td>40% (w/v) acrylamide solution</td>
			<td>Nacalai Tesque</td>
			<td>06119-45</td>
			<td>Acrylamide:Bis = 29:1</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Nacalai Tesque</td>
			<td>33401-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Wako</td>
			<td>016-08021</td>
			<td>10% (w/v) solution was prepared by dissolving the powder of ammonium persulfate in MilliQ water</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>166-04831</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese chloride tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M3634</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Prestained Standard</td>
			<td>Bio-Rad</td>
			<td>1610374, 1610373, 1610377</td>
			<td>Molecular weight marker used in the protocol</td>
		</tr>
		<tr>
			<td>WIDE-VIEW Prestained Protein Size Marker III</td>
			<td>Wako</td>
			<td>230-02461</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Nacalai Tesque</td>
			<td>17109-64</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Protran NC 0.45</td>
			<td>GE Healthcare</td>
			<td>10600007</td>
			<td>Nitrocellulose membrane</td>
		</tr>
		<tr>
			<td>Durapore Membrane Filter</td>
			<td>EMD Millipore</td>
			<td>GVHP00010</td>
			<td>PVDF membrane</td>
		</tr>
		<tr>
			<td>Filter Papers No.1</td>
			<td>Advantec</td>
			<td>00013600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S</td>
			<td>Nacalai Tesque</td>
			<td>28322-72</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Wako</td>
			<td>017-00251</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td>polyoxyethylenesorbitan monolaurate</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Wako</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Difco Laboratories</td>
			<td>232100</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunostar</td>
			<td>Wako</td>
			<td>291-55203</td>
			<td>ECL solution (Normal sensitivity)</td>
		</tr>
		<tr>
			<td>Immunostar LD</td>
			<td>Wako</td>
			<td>290-69904</td>
			<td>ECL solution (High sensitivity)</td>
		</tr>
		<tr>
			<td>CBB staining solution</td>
			<td>homemade</td>
			<td></td>
			<td>1 g CBB R-250, 50% (v/v) methanol, 10% (v/v) acetic acid in 1 L of MilliQ water</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td></td>
		</tr>
		<tr>
			<td>CBB destaining solution</td>
			<td>homemade</td>
			<td></td>
			<td>12% (v/v) methanol, 7% (v/v) acetic acid in 1 L MilliQ water</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-HA antibody</td>
			<td>Sigma-Aldrich</td>
			<td>11583816001</td>
			<td>Used at 0.2 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-Rab10 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>#8127</td>
			<td>Used at 1:1000 for immunoblotting. 
			Specificity was confirmed by CRISPR KO in Ito et al., Biochem J, 2016.</td>
		</tr>
		<tr>
			<td>anti-pSer935 antibody</td>
			<td>Abcam</td>
			<td>ab133450</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-LRRK2 antibody</td>
			<td>Abcam</td>
			<td>ab133518</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-&#945;-tubulin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>T9026</td>
			<td>Used at 1 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>anti-GAPDH antibody</td>
			<td>Santa-Cruz</td>
			<td>sc-32233</td>
			<td>Used at 0.02 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Sheep Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>515-035-003</td>
			<td>Used at 0.16 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-003</td>
			<td>Used at 0.16 &#956;g/mL for immunoblotting.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Inhibitors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSK2578215A</td>
			<td>MedChem Express</td>
			<td>HY-13237</td>
			<td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>MLi-2</td>
			<td>Provided by Dr Dario Alessi (University of Dundee)</td>
			<td></td>
			<td>Stock solution was prepared in DMSO at 10 mM and stored at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rab10/pcDNA5 FRT TO HA</td>
			<td>Provided by Dr Dario Alessi 
			(University of Dundee)</td>
			<td></td>
			<td>This plasmid expresses amino-terminally HA-tagged human Rab10.</td>
		</tr>
		<tr>
			<td>LRRK2 WT/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Ito et al., Biochemistry, 46: 1380&#8211;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged wild-type human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 K1906M/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Ito et al., Biochemistry, 46: 1380&#8211;1388 (2007). This plasmid expresses amino-terminally 3xFLAG-tagged K1906M kinase-inactive mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 N1437H/p3xFLAG-CMV-10</td>
			<td></td>
			<td></td>
			<td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged N1437H FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441C/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441C FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441G/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441G FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441H/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged R1441H FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 R1441S/p3xFLAG-CMV-10</td>
			<td></td>
			<td></td>
			<td>This paper. This plasmid expresses amino-terminally 3xFLAG-tagged R1441S FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 Y1699C/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged Y1699C FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 G2019S/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged G2019S FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>LRRK2 I2020T/p3xFLAG-CMV-10</td>
			<td>Provided by Dr Takeshi Iwatsubo (University of Tokyo)</td>
			<td></td>
			<td>Kamikawaji et al., Biochemistry, 48: 10963&#8211;10975 (2013). This plasmid expresses amino-terminally 3xFLAG-tagged I2020T FPD mutant of human LRRK2.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Forma Series II 3110 Water-Jacketed</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Pipette</td>
			<td>Drummond</td>
			<td>Pipet-Aid PA-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette P10</td>
			<td>Nichiryo</td>
			<td>00-NPX2-10</td>
			<td>0.5&#8211;10 &#956;L</td>
		</tr>
		<tr>
			<td>Micropipette P200</td>
			<td>Nichiryo</td>
			<td>00-NPX2-200</td>
			<td>20&#8211;200 &#956;L</td>
		</tr>
		<tr>
			<td>Micropipette P1000</td>
			<td>Nichiryo</td>
			<td>00-NPX2-1000</td>
			<td>100&#8211;1000 &#956;L</td>
		</tr>
		<tr>
			<td>Tips for micropipette P10</td>
			<td>STAR</td>
			<td>RST-481LCRST</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Tips for micropipette P200</td>
			<td>FUKAEKASEI</td>
			<td>1201-705YS</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Tips for micropipette P1000</td>
			<td>STAR</td>
			<td>RST-4810BRST</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>5 mL disporsable pipette</td>
			<td>Greiner</td>
			<td>606180</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>10 mL disporsable pipette</td>
			<td>Greiner</td>
			<td>607180</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>25 mL disporsable pipette</td>
			<td>Falcon</td>
			<td>357535</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Hematocytometer</td>
			<td>Sunlead Glass</td>
			<td>A126</td>
			<td>Improved Neubeuer</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>CKX53</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>AGC Techno Glass</td>
			<td>3810-006</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers</td>
			<td>Sumitomo Bakelite</td>
			<td>MS-93100</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>STAR</td>
			<td>RSV-MTT1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>AGC Techno Glass</td>
			<td>2323-015</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tubes</td>
			<td>AGC Techno Glass</td>
			<td>2343-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuges</td>
			<td>TOMY</td>
			<td>MX-307</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greiner</td>
			<td>655061</td>
			<td>Not for tissue culture</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M2e</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS&#8211;PAGE tanks</td>
			<td>Nihon Eido</td>
			<td>NA-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer tanks</td>
			<td>Nihon Eido</td>
			<td>NA-1510B</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel plates (notched)</td>
			<td>Nihon Eido</td>
			<td>NA-1000-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel plates (plain)</td>
			<td>Nihon Eido</td>
			<td>NA-1000-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon spacers</td>
			<td>Nihon Eido</td>
			<td>NA-1000-16</td>
			<td></td>
		</tr>
		<tr>
			<td>17-well combs</td>
			<td>Nihon Eido</td>
			<td>Custom made</td>
			<td></td>
		</tr>
		<tr>
			<td>Binder clips</td>
			<td>Nihon Eido</td>
			<td>NA-1000-15</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Terumo</td>
			<td>SS-05SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>21G</td>
			<td>Terumo</td>
			<td>NN-2138R</td>
			<td></td>
		</tr>
		<tr>
			<td>Power Station 1000 VC</td>
			<td>ATTO</td>
			<td>AE-8450</td>
			<td>Power supply for SDS&#8211;PAGE and transfer</td>
		</tr>
		<tr>
			<td>Large weighing boats</td>
			<td>Ina Optika</td>
			<td>AS-DL</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic containers</td>
			<td>AS ONE</td>
			<td>PS CASE No.4</td>
			<td>10 x 80 x 50 mm</td>
		</tr>
		<tr>
			<td>Rocking shaker</td>
			<td>Titech</td>
			<td>NR-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrene foam box</td>
			<td>generic</td>
			<td></td>
			<td>The internal dimensions should fit one transfer tank (200 x 250 x 250 mm).</td>
		</tr>
		<tr>
			<td>ImageQuant LAS-4000</td>
			<td>GE Healthcare</td>
			<td></td>
			<td>An imager equipped with a cooled CCD camera for detection of ECL</td>
		</tr>
	</tbody>
</table>

rab10 phosphorylation detection by lrrk2 activity using sds-page with a phosphate-binding tag

Mutationer i leucin-rig gentage kinase 2 (LRRK2) har vist sig at være forbundet med familiær Parkinsons sygdom (FPD). Da unormal aktivering af kinase aktiviteten af LRRK2 har været involveret i patogenesen af PD, er det vigtigt at fastlægge en metode til at evaluere de fysiologiske niveauer af kinase aktiviteten i LRRK2. Nylige undersøgelser afslørede, at LRRK2 phosphorylates Rab GTPase familiemedlemmer, herunder Rab10, fysiologiske betingelser. Selvom fosforylering af endogent Rab10 ved LRRK2 i kulturperler celler kunne påvises ved massespektrometri, har det været vanskeligt at påvise det ved immunoblotting på grund af den ringe følsomhed af foreliggende fosforylering-specifikke antistoffer for Rab10. Her, vi beskriver en simpel metode til påvisning af de endogene niveauer af Rab10 fosforylering af LRRK2 baseret på immunoblotting udnytte sodium dodecyl sulfat polyacrylamid gelelektroforese (SDS-PAGE) kombineret med en fosfat-bindende tag (P-tag), som er N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl) -Nielsen,N',N'- tris (pyridin-2-yl-methyl) - 1,3 - diaminopropan-2-ol. Denne protokol ikke kun giver et eksempel på den metode, der udnytter P-tag, men giver også mulighed for at vurdere hvordan mutationer samt hæmmer behandling/administration eller nogen andre faktorer ændre downstream signalering af LRRK2 i celler og væv .

Overekspression System: Fosforylering af HA-Rab10 af 3 × FLAG-LRRK2 i HEK293 celler:
HEK293 celler blev transfekteret med 0.266 µg af HA-Rab10 wild-type og 1.066 µg af 3 × FLAG-LRRK2 (wild-type, kinase-inaktive mutant (K1906M), eller FPD mutanter). Rab10 fosforylering blev undersøgt af P-tag SDS-PAGE efterfulgt af immunoblotting ved hjælp af en anti-HA antistof (figur 2). 10 µg af proteiner blev...

Watch this Scientific Journal Video about Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag at JoVE.com

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Den nuværende undersøgelse beskriver en simpel metode til påvisning af endogene niveauer af Rab10 fosforylering af leucin-rig gentage kinase 2.

Rab10 fosforylering påvisning af LRRK2 aktivitet ved hjælp af SDS-PAGE med en fosfat-bindende Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Research

JoVE Journal

Biochemistry

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Meget følsomme og Rapid Fluorescens Detection med en bærbar FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Påvisning af pH-afhængige aktivitet af<em&gt; Escherichia coli</em&gt; Chaperone HdeB<em&gt; In vitro</em&gt; og<em&gt; In vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Assaying Protein Kinase Activity with Radiolabeled ATP

Analyse af proteinkinaseaktivitet med radiomærket ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

DNA Polymerase aktivitet Assay ved hjælp af nær-infrarødt fluorescerende mærket DNA visualiseret af acrylamid Gel elektroforese

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

Påvisning af små GTPase Prenylation og GTP bindende ved hjælp af membran fraktionering og GTPase-forbundet Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identifikation af Cyclin-afhængig Kinase 1 specifikke fosforylering websteder ved en In Vitro -Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Affinitet rensning af grønkorn Translocon Protein komplekser ved hjælp af tryk på Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Human perifert blod Neutrofile Isolation for at forhøre Parkinsons Associated LRRK2 Kinase Pathway ved at vurdere Rab10 fosforylering

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Påvisning af proteinubiquitinationssteder ved peptidberigelse og massespektrometri

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetisk screening af Nukleaseaktivitet ved hjælp af nukleinsyre sonder

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...