Name: Enzymatische modificatie en beoordeling van flowcytometrie van door het gistoppervlak weergegeven eiwitten (Video) | JoVE Journal
Uploaded: 2025-05-30T14:30:00.000+00:00
Duration: 10 min 54 s
Description: Leer enzymatische modificatie en flowcytometrie-evaluatie van gistoppervlak-weergegeven eiwitten. Deze methode maakt kinetische mapping van enzymatische reacties mogelijk voor onderzoek naar eiwitchemische modificaties.

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<ol><li>Lea, M. A., Weber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+enzymes+in+homeostasis%3A+VIII.+Inhibition+of+the+activity+of+glycolytic+enzymes+by+free+fatty+acids%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Role of enzymes in homeostasis: VIII. Inhibition of the activity of glycolytic enzymes by free fatty acids.</a> J Biol Chem. 243 (6), 1096-1102 (1968).</li>
<li>Mahé, M., Rios-Fuller, T. J., Karolin, A., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+enzymatic+dysfunctions+in+metabolic+disorders+and+cancer%5BTitle%5D)+AND+%22Front+Oncol%22%5BJournal%5D)">Genetics of enzymatic dysfunctions in metabolic disorders and cancer.</a> Front Oncol. 13, 1230934(2023).</li>
<li>Fernandez-de-Cossio-Diaz, J., Vazquez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+physical+model+of+cell+metabolism%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">A physical model of cell metabolism.</a> Sci Rep. 8 (1), 8349(2018).</li>
<li>Metallo, C. M., Vander Heiden, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+metabolic+regulation+and+its+influence+on+cell+physiology%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Understanding metabolic regulation and its influence on cell physiology.</a> Mol Cell. 49 (3), 388-398 (2013).</li>
<li>Mildvan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+signaling+and+related+enzymes%5BTitle%5D)+AND+%22Proteins%22%5BJournal%5D)">Mechanisms of signaling and related enzymes.</a> Proteins. 29 (4), 401-416 (1997).</li>
<li>Frosina, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpression+of+enzymes+that+repair+endogenous+damage+to+DNA%5BTitle%5D)+AND+%22Eur+J+Biochem%22%5BJournal%5D)">Overexpression of enzymes that repair endogenous damage to DNA.</a> Eur J Biochem. 267 (8), 2135-2149 (2000).</li>
<li>Schärer, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemistry+and+biology+of+DNA+repair%5BTitle%5D)+AND+%22Angew+Chem+Int+Ed%22%5BJournal%5D)">Chemistry and biology of DNA repair.</a> Angew Chem Int Ed. 42 (26), 2946-2974 (2003).</li>
<li>de la Fuente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme+therapy%3A+Current+challenges+and+future+perspectives%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Enzyme therapy: Current challenges and future perspectives.</a> Int J Mol Sci. 22 (17), 9181(2021).</li>
<li>Robertson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzymes+as+a+special+class+of+therapeutic+target%3A+clinical+drugs+and+modes+of+action%5BTitle%5D)+AND+%22Curr+Opin+Struct+Biol%22%5BJournal%5D)">Enzymes as a special class of therapeutic target: clinical drugs and modes of action.</a> Curr Opin Struct Biol. 17 (6), 674-679 (2007).</li>
<li>Goddard, J. -P., Reymond, J. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme+assays+for+high-throughput+screening%5BTitle%5D)+AND+%22Curr+Opin+Biotechnol%22%5BJournal%5D)">Enzyme assays for high-throughput screening.</a> Curr Opin Biotechnol. 15 (4), 314-322 (2004).</li>
<li>Helm, J. S., Hu, Y., Chen, L., Gross, B., Walker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+active-site+inhibitors+of+MurG+using+a+generalizable%2C+high-throughput+glycosyltransferase+screen%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">Identification of active-site inhibitors of MurG using a generalizable, high-throughput glycosyltransferase screen.</a> J Am Chem Soc. 125 (37), 11168-11169 (2003).</li>
<li>Veldhuyzen, W. F., Nguyen, Q., McMaster, G., Lawrence, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+light-activated+probe+of+intracellular+protein+kinase+activity%5BTitle%5D)+AND+%22J+Am+Chem+Soc%22%5BJournal%5D)">A light-activated probe of intracellular protein kinase activity.</a> J Am Chem Soc. 125 (44), 13358-13359 (2003).</li>
<li>Torres, M., Forman, H. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTorres%2C+M+%22Encyclopedia+of+Respiratory%22">Encyclopedia of Respiratory</a>. Laurent, G. J., Shapiro, S. D. , Academic Press. 10-18 (2006).</li>
<li>Blume-Jensen, P., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oncogenic+kinase+signalling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Oncogenic kinase signalling.</a> Nature. 411 (6835), 355-365 (2001).</li>
<li>Martin, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+signaling+and+cancer%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Cell signaling and cancer.</a> Cancer Cell. 4 (3), 167-174 (2003).</li>
<li>Lothrop, A. P., Torres, M. P., Fuchs, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deciphering+post-translational+modification+codes%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Deciphering post-translational modification codes.</a> FEBS Lett. 587 (8), 1247-1257 (2013).</li>
<li>Graves, J. D., Krebs, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+phosphorylation+and+signal+transduction%5BTitle%5D)+AND+%22Pharmacol+Ther%22%5BJournal%5D)">Protein phosphorylation and signal transduction.</a> Pharmacol Ther. 82 (2), 111-121 (1999).</li>
<li>Hafen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinases+and+phosphatases--A+marriage+is+consummated%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Kinases and phosphatases--A marriage is consummated.</a> Science. 280 (5367), 1212-1213 (1998).</li>
<li>Westphal, R. S., Anderson, K. A., Means, A. R., Wadzinski, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+signaling+complex+of+Ca2%2B-calmodulin-dependent+protein+kinase+IV+and+protein+phosphatase+2A%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A signaling complex of Ca2+-calmodulin-dependent protein kinase IV and protein phosphatase 2A.</a> Science. 280 (5367), 1258-1261 (1998).</li>
<li>Barford, D., Das, A. K., Egloff, M. -P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+structure+and+mechanism+of+protein+phosphatases%3A+Insights+into+catalysis+and+regulation%5BTitle%5D)+AND+%22Annu+Rev+Biophys+Biomol+Struct%22%5BJournal%5D)">The structure and mechanism of protein phosphatases: Insights into catalysis and regulation.</a> Annu Rev Biophys Biomol Struct. 27, 133-164 (1998).</li>
<li>Liu, Q., Qu, J., Zhao, M., Xu, Q., Sun, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+SHP2+as+a+promising+strategy+for+cancer+immunotherapy%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">Targeting SHP2 as a promising strategy for cancer immunotherapy.</a> Pharmacol Res. 152, 104595(2020).</li>
<li>Pan, J., Zhou, L., Zhang, C., Xu, Q., Sun, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+protein+phosphatases+for+the+treatment+of+inflammation-related+diseases%3A+From+signaling+to+therapy%5BTitle%5D)+AND+%22Signal+Transduct+Targeted+Ther%22%5BJournal%5D)">Targeting protein phosphatases for the treatment of inflammation-related diseases: From signaling to therapy.</a> Signal Transduct Targeted Ther. 7 (1), 177(2022).</li>
<li>Denard, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((YESS+2.0%2C+a+tunable+platform+for+enzyme+evolution%2C+yields+highly+active+TEV+protease+variants%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">YESS 2.0, a tunable platform for enzyme evolution, yields highly active TEV protease variants.</a> ACS Synth Biol. 10 (1), 63-71 (2021).</li>
<li>Lim, S., Glasgow, J. E., Filsinger Interrante, M., Storm, E. M., Cochran, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dual+display+of+proteins+on+the+yeast+cell+surface+simplifies+quantification+of+binding+interactions+and+enzymatic+bioconjugation+reactions%5BTitle%5D)+AND+%22Biotechnol+J%22%5BJournal%5D)">Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions.</a> Biotechnol J. 12 (5), (2017).</li>
<li>Yi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+of+TEV+protease+variants+by+yeast+ER+sequestration+screening+%28YESS%29+of+combinatorial+libraries%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries.</a> Proc Natl Acad Sci USA. 110 (18), 7229-7234 (2013).</li>
<li>Yi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yeast+endoplasmic+reticulum+sequestration+screening+for+the+engineering+of+proteases+from+libraries+expressed+in+yeast%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Yeast endoplasmic reticulum sequestration screening for the engineering of proteases from libraries expressed in yeast.</a> Methods Mol Biol. 1319, 81-93 (2015).</li>
<li>Semenza, J. C., Hardwick, K. G., Dean, N., Pelham, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ERD2%2C+a+yeast+gene+required+for+the+receptor-mediated+retrieval+of+luminal+ER+proteins+from+the+secretory+pathway%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway.</a> Cell. 61 (7), 1349-1357 (1990).</li>
<li>Mei, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+aromatic+residue-controlled+protein+retention+in+the+endoplasmic+reticulum+of+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Characterization of aromatic residue-controlled protein retention in the endoplasmic reticulum of Saccharomyces cerevisiae.</a> J Biol Chem. 292 (50), 20707-20719 (2017).</li>
<li>Ezagui, J., Russell, B., Mairena, Y., Stern, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endoplasmic+reticulum+sequestration+empowers+phosphorylation+profiling+on+the+yeast+surface%5BTitle%5D)+AND+%22AIChE+J%22%5BJournal%5D)">Endoplasmic reticulum sequestration empowers phosphorylation profiling on the yeast surface.</a> AIChE J. 68 (12), e17931(2022).</li>
<li>Kawai, S., Murata, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKawai%2C+S+%22Genetic+Transformation+Systems+in+Fungi%22">Genetic Transformation Systems in Fungi</a>. van den Berg, M. A., Maruthachalam, K. 1, Springer International Publishing. 187-192 (2015).</li>
<li>Kawai, S., Hashimoto, W., Murata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transformation+of+Saccharomyces+cerevisiae+and+other+fungi%3A+methods+and+possible+underlying+mechanism%5BTitle%5D)+AND+%22Bioeng+Bugs%22%5BJournal%5D)">Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism.</a> Bioeng Bugs. 1 (6), 395-403 (2010).</li>
<li>Loock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-efficiency+transformation+and+expression+of+genomic+libraries+in+yeast%5BTitle%5D)+AND+%22Methods+Protoc%22%5BJournal%5D)">High-efficiency transformation and expression of genomic libraries in yeast.</a> Methods Protoc. 6 (5), 89(2023).</li>
<li>Huang, D., Gore, P. R., Shusta, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+yeast+secretion+of+heterologous+proteins+by+regulating+expression+rates+and+post-secretory+loss%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss.</a> Biotechnol Bioeng. 101 (6), 1264-1275 (2008).</li>
<li>Yu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+protein+tyrosine+phosphatase+SHP2+for+the+treatment+of+PTPN11-associated+malignancies%5BTitle%5D)+AND+%22Mol+Cancer+Ther%22%5BJournal%5D)">Targeting protein tyrosine phosphatase SHP2 for the treatment of PTPN11-associated malignancies.</a> Mol Cancer Ther. 12 (9), 1738-1748 (2013).</li>
<li>Stern, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Geometry+and+expression+enhance+enrichment+of+functional+yeast-displayed+ligands+via+cell+panning%5BTitle%5D)+AND+%22Biotechnol+Bioeng%22%5BJournal%5D)">Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning.</a> Biotechnol Bioeng. 113 (11), 2328-2341 (2016).</li>
<li>Pan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+single-cell+gates+for+yeast+display+screening%5BTitle%5D)+AND+%22Protein+Eng+Design+Sel%22%5BJournal%5D)">Optimized single-cell gates for yeast display screening.</a> Protein Eng Design Sel. 38, gzae018(2025).</li>
<li>Margittai, É, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+H2O2+in+the+endoplasmic+reticulum+promotes+in+vivo+disulfide+bond+formation%5BTitle%5D)+AND+%22Antioxid+Redox+Signal%22%5BJournal%5D)">Production of H2O2 in the endoplasmic reticulum promotes in vivo disulfide bond formation.</a> Antioxid Redox Signal. 16 (10), 1088-1099 (2012).</li>
<li>Werner-Washburne, M., Braun, E., Johnston, G. C., Singer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stationary+phase+in+the+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Microbiol+Rev%22%5BJournal%5D)">Stationary phase in the yeast Saccharomyces cerevisiae.</a> Microbiol Rev. 57 (2), 383-401 (1993).</li>
<li>Ezagui, J., Stern, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tyrosine+phosphorylation+screening+on+the+yeast+surface+by+magnetic+bead+selection+and+FACS%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Tyrosine phosphorylation screening on the yeast surface by magnetic bead selection and FACS.</a> Methods Mol Biol. 2681, 275-290 (2023).</li>
<li>Yarnall, M. T. N., Kim, S. H., Korntner, S., Bishop, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Destabilization+of+the+SHP2+and+SHP1+protein+tyrosine+phosphatase+domains+by+a+non-conserved+%E2%80%9Cbackdoor%E2%80%9D+cysteine%5BTitle%5D)+AND+%22Biochem+Biophys+Rep%22%5BJournal%5D)">Destabilization of the SHP2 and SHP1 protein tyrosine phosphatase domains by a non-conserved “backdoor” cysteine.</a> Biochem Biophys Rep. 32, 101370(2022).</li>
<li>Dustin, C. M., Heppner, D. E., Lin, M. J., van der Vliet, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redox+regulation+of+tyrosine+kinase+signalling%3A+more+than+meets+the+eye%5BTitle%5D)+AND+%22J+Biochem%22%5BJournal%5D)">Redox regulation of tyrosine kinase signalling: more than meets the eye.</a> J Biochem. 167 (2), 151-163 (2020).</li>
<li>Stern, L. A., Csizmar, C. M., Woldring, D. R., Wagner, C. R., Hackel, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Titratable+avidity+reduction+enhances+affinity+discrimination+in+mammalian+cellular+selections+of+yeast-displayed+ligands%5BTitle%5D)+AND+%22ACS+Comb+Sci%22%5BJournal%5D)">Titratable avidity reduction enhances affinity discrimination in mammalian cellular selections of yeast-displayed ligands.</a> ACS Comb Sci. 19 (5), 315-323 (2017).</li>
<li>Waldman, A. C., Rao, B. M., Keung, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+the+residue+specificities+of+epigenome+enzymes+by+yeast+surface+display%5BTitle%5D)+AND+%22Cell+Chem+Biol%22%5BJournal%5D)">Mapping the residue specificities of epigenome enzymes by yeast surface display.</a> Cell Chem Biol. 28 (12), 1772-1779.e4 (2021).</li>
</ol>

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enzymatische modificatie en beoordeling van flowcytometrie van door het gistoppervlak weergegeven eiwitten

Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

Enzymatische modificatie en beoordeling van flowcytometrie van door het gistoppervlak weergegeven eiwitten

Yeast surface display is a genotype-phenotype linkage strategy that empowers high-throughput screening of protein function. Traditionally, yeast surface ...

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Research

JoVE Journal

Bioengineering

Bio-engineering

Een robuuste benadering van de levering van organellen monitor aan op de zure lumen van de gist vacuole voor afbraak en recycling wordt beschreven. De methode is gebaseerd op de specifieke etikettering van target organellen met een genetisch gecodeerd dual-emissie fluorescentie pH-biosensor, en visualisatie van individuele cellen met behulp van fluorescentie microscopie.

Een fluorescentie microscopie Assay voor de bewaking Mitophagy in de Gist Saccharomyces cerevisiae

Kwantitatieve Live Cell Fluorescentie-microscopie analyse van Fission Yeast

Twee aanvullende methoden op basis van flowcytometrie en microscopie gepresenteerd die de kwantificering van de enkele cel, de dynamiek van genexpressie geïnduceerd door de activering van MAPK route in gist mogelijk.

Temporele Kwantificering van MAPK geïnduceerde expressie in Single gistcellen

Dit artikel beschrijft een op groei gebaseerde gisttest voor de bepaling van genetische vereisten voor eiwitafbraak. Het demonstreert ook een methode voor snelle extractie van gisteiwitten, geschikt voor western blotting om biochemisch de afbraakvereisten te bevestigen. Deze technieken kunnen worden aangepast om de afbraak van een verscheidenheid aan eiwitten te monitoren.

-Groei op basis Vaststelling en Biochemische Bevestiging van Genetische Eisen voor de afbraak van eiwitten in<em&gt; Saccharomyces cerevisiae</em

Accurate kwantificering van vesiculaire transportgebeurtenissen levert vaak sleutelinzichten op in de rol van specifieke eiwitten en de effecten van mutaties. Dit artikel presenteert methoden voor het gebruik van superecliptic pHluorin, een pH-gevoelige GFP-variant, als een hulpmiddel voor kwantificering van endocytische gebeurtenissen in levende cellen met behulp van kwantitatieve fluorescentiemicroscopie en flowcytometrie.

Toepassingen van pHluorin voor Quantitative, Kinetic en High-throughput analyse van de Humor in gist

Dit artikel beschrijft een FRET-based flow cytometrie protocol voor het kwantificeren van eiwit zelf montage in zowel S. cerevisiae en HEK293T cellen.

Opsporen en karakteriseren van zelf-assemblage van eiwitten in vivo door flow Cytometrie

Splijtingsgist wordt hier gebruikt als een heterologe gastheer om bacteriële cytoskeletale eiwitten zoals FtsZ en MreB tot expressie te brengen als translationele fusie-eiwitten met GFP om hun polymerisatie te visualiseren. Ook worden verbindingen die de polymerisatie beïnvloeden geïdentificeerd door beeldvorming met behulp van een fluorescentiemicroscoop.

Splijtingsgist als platform voor antibacteriële geneesmiddelenschermen gericht op bacteriële cytoskeleteiwitten

Dit protocol beschrijft de essentiële stappen voor het uitvoeren van selectiecampagnes voor gistoppervlakken om eiwitvarianten te verrijken die binden aan een interessant antigeen.

Eiwittechniek door gist Surface Display

Hier presenteren we een protocol om de overleving na stress in gistmonsters te kwantificeren. De test maakt gebruik van twee fluorescerende kleurstoffen, SYTO 9 en propidiumjodide (PI), om de integriteit van het plasmamembraan te kwantificeren. Het maakt gebruik van flowcytometrie om kwantitatieve en reproduceerbare schattingen te geven van de levende, dode en beschadigde celfracties voor monsters na oxidatieve stress.

Een geoptimaliseerde LIVE/DEAD-test in combinatie met flowcytometrie voor het kwantificeren van overleving na stress in gistcellen

In dit artikel beschrijven we een methode gebruik te maken van multi-spectraal imaging flowcytometrie om de internalisering van polyanhydride nanodeeltjes of bacteriën door RAW 264.7 cellen te kwantificeren.

Het analyseren van cellulaire internalisatie van nanodeeltjes en bacteriën door Multi-spectrale Imaging flowcytometrie

Leer enzymatische modificatie en flowcytometrie-evaluatie van gistoppervlak-weergegeven eiwitten. Deze methode maakt kinetische mapping van enzymatische reacties mogelijk voor onderzoek naar eiwitchemische modificaties.

Enzymatische modificatie en beoordeling van flowcytometrie van door het gistoppervlak weergegeven eiwitten

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