diplomsko delo univerzitetnega študijskega programa I. stopnje
Dea Simonič (Avtor), Uroš Potočnik (Mentor), Larisa Goričan (Komentor)

Povzetek

Mononuklearne celice periferne krvi (MCPK) so ključna komponenta imunskega sistema za boj proti infekcijam in prilagajanje patogenom. MCPK se nahajajo v periferni venski krvi in predstavljajo lahko dostopen vir imunskih celic za nadaljnje imunske in toksikološke raziskave, pomembne so pri analizi genske in proteinske ekspresije v krvi, proteini MCPK pa predstavljajo pomembne biomarkerje imunskih in možganskožilnih bolezni. V večini standardnih postopkov lize celice se za tvorbo celičnega lizata uporablja en sam lizni pufer, kar lahko vodi do nespecifičnih interakcij proteinov, raztapljanja proteinov v pufru, ki jih ne želimo analizirati, prav tako so možne težave s topnostjo proteina v pufru. Analiza proteinov s subceličnim frakcioniranjem (s sekvenčno uporabo več liznih pufrov) pa med drugim poveča možnosti, da je željeni protein izoliran in bistveno zmanjša možnost kontaminacije z drugimi proteini. Subcelično frakcioniranje prav tako omogoča pridobitev informacije o tem kako se porazdelitev posameznih proteinov razlikuje med različnimi subceličnimi kompartmenti, celicami in tkivi, ter daje informacijo o stanju celic in o aktivaciji signalnih poti. V diplomski nalogi smo optimizirali protokol za izolacijo proteinov s subcelično frakcionacijo iz svežih MCPK, povzet iz članka Holden P., Horton W. A.: Crude subcellular fractions of cultured mammalian cell lines, ki opisuje optimizacijo protokola za gojene celične linije sesalcev. V sklopu naloge smo optimizirali koncentracijo digitonina v digitonin pufru in volumne ostalih pufrov za subcelično frakcionacijo. Optimiziran protokol za izolacijo proteinov s subcelično frakcionacijo svežih MCPK smo primerjali z izolacijo proteinov z dodatkom enega samega liznega pufra ter izbrane optimizirane pogoje preverili tudi na zamrznjenih MCPK. Vse izolirani proteine smo analizirali z metodo western prenosa (WB) in rezultate kvantificirali s programom ImageLab. S testiranjem različnih koncentracij digitonina v digitonin pufru in na podlagi WB analize za proteinske markerje α-tubulin, citratna sintaza (CS) in histonska deacetilaza 6 (HDA-C6) smo ugotovili, da je optimalna koncentracija digitonina v digitonin pufru, za izolacijo le citosolnih proteinov svežih vzorcev MCPK, 50 μg/mL. Z optimizacijo koncentracije digitonina in volumnov dodanih liznih pufrov smo ločeno izolirali citosolne proteine, proteine membranskih organelov, jedra in netopnih proteinov. Ugotovili smo, da je izplen izoliranih proteinov pri izolaciji s subcelično frakcionacijo večji kot pri direktni izolaciji proteinov z dodatkom enega samega liznega pufra.

Ključne besede

MCPK;subcelična frakcionacija;izolacija proteinov iz različnih celičnih kompartmentov;lizni pufri;western prenos;diplomske naloge;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.11 - Diplomsko delo
Organizacija: UM FKKT - Fakulteta za kemijo in kemijsko tehnologijo
Založnik: [D. Simonič]
UDK: 576.32/.36(043.2)
COBISS: 77660675 Povezava se bo odprla v novem oknu
Št. ogledov: 530
Št. prenosov: 67
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Subcellular fractionation of human peripheral blood mononuclear cells and isolation of proteins from different cellular compartments
Sekundarni povzetek: Peripheral blood mononuclear cells (PBMC) are an important immune system component for fighting infections and adapting to pathogens. PBMCs are located in peripheral venous blood, and they represent an easily accessible source of immune cells for further immune and toxicological research, they represent important biomarkers of immune and cerebrovascular diseases and allow analyzes of gene and protein expression from the blood. Most standard procedures use a single lysis buffer for cell lysis, leading to non-specific protein interaction, dissolution of proteins in the buffer that we don't want to analyze, and potential problems with protein solubility. Analysis of proteins by subcellular fractionation (by sequential use of multiple lysis buffers) increases the chances of isolating the protein of interest and lowers the chances of contamination with unwanted proteins. Subcellular fractionation also allows for obtaining information about the cell condition and signaling pathways activation status. In the thesis, we have optimized the protocol for the isolation of proteins by subcellular fractionation from fresh PBMC. The protocol was adapted from the article Holden P., Horton W. A.: Crude subcellular fractions of cultured mammalian cell lines, describing the optimization of the isolation protocol for cultured mammalian cell lines. In the thesis, we optimized the digitonin concentration in the digitonin-containing buffer and the volumes of the other three buffers used for subcellular fractionation. The optimized protein isolation with subcellular fractionation of fresh PBMC was then compared with protein isolation using only a single lysis buffer. Also, the selected optimized conditions were tested on frozen PBMC. All isolated proteins were analyzed by the Western Blot (WB) method, and the results were quantified with the ImageLab software. By testing diffrent concentrations of digitonin in digitonin buffer and based on WB analysis for protein markers α-tubulin, citrate synthase (CS) and histone deacetylase 6 (HDA-C6), we found that the optimal concentration of digitonin in digitonin buffer, for isolation of only cytosolic proteins from fresh PBMC samples, is 50 μg/mL. By optimizing digitonin concentration and the volumes of added lysis buffers, cytosolic proteins, membrane organelle proteins, nuclei and insoluble proteins were isolated seperately. We found that the yield of isolated proteins by subcellular fractionation was higher than in direct protein isolation with the addition of a single lysis buffer.
Sekundarne ključne besede: PBMC;subcellular fractionation;isolation of proteins from different cellular compartments;lysis buffers;Western Blot;
Vrsta dela (COBISS): Diplomsko delo/naloga
Komentar na gradivo: Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Strani: XII, 53 str.
ID: 13295249