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master's thesis
Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom
Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry, 2016. urn:nbn:hr:163:993535
Radić, Dora
University of Zagreb
Faculty of Pharmacy and Biochemistry
Department of medical biochemistry and haematology

Institutional repository: Repository of Faculty of Pharmacy and Biochemistry University of Zagreb

Cite this document

Radić, D. (2016). Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom (Master's thesis). Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry. Retrieved from https://urn.nsk.hr/urn:nbn:hr:163:993535

Radić, Dora. "Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2016. https://urn.nsk.hr/urn:nbn:hr:163:993535

Radić, Dora. "Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom." Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, 2016. https://urn.nsk.hr/urn:nbn:hr:163:993535

Radić, D. (2016). 'Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom', Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, accessed 02 May 2024, https://urn.nsk.hr/urn:nbn:hr:163:993535

Radić D. Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom [Master's thesis]. Zagreb: University of Zagreb, Faculty of Pharmacy and Biochemistry; 2016 [cited 2024 May 02] Available at: https://urn.nsk.hr/urn:nbn:hr:163:993535

D. Radić, "Optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom", Master's thesis, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, 2016. Available at: https://urn.nsk.hr/urn:nbn:hr:163:993535

Metadata
TitleOptimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK Western blot metodom
Title (english)Optimizing conditions for determination of the signaling molecules ERK and p38 MAPK by Western blot analysis
AuthorDora Radić
MentorLada Rumora (mentor)
Committee memberLada Rumora (predsjednik povjerenstva)
Committee memberJasmina Lovrić (član povjerenstva)
Committee memberMarija Grdić Rajković (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Pharmacy and Biochemistry
(Department of medical biochemistry and haematology)
Zagreb
Defense date and country2016-07-01, Croatia
Scientific / art field,
discipline and subdiscipline		BIOMEDICINE AND HEALTHCARE
Pharmacy
Pharmacy
Abstract
Kronična opstrukcijska plućna bolest karakterizirana je snažnim upalnim odgovorom na inhalaciju štetnih čestica. Dolazi do nakupljanja imunosnih stanica koje luče citokine, kemokine i tkivne proteaze, posljedično tome dolazi do opstrukcije dišnih putova i izazito smanjenog protoka zraka. TLR receptori odgovorni su prvenstveno za urođenu imunost i za pojačavanje odgovora stečenog imuniteta. Nađena je veza između aktivacije TLR receptora, pokretanja kaskadne reakcije kojom dolazi do aktivacije (foforilacije) MAPK kinaza (ERK, p38, JNK) i izlučivanja različitih citokina koji izazivaju upalni odgovor. Cigaretni dim te brojni drugi štetni čimbenici iz okoliša dovode do aktivacije TLR receptora. Agonist TLR4 (LPS) i agonist TLR2 (LTA) koriste se u svrhu ipitivanja upalnog odgovora potaknutog navedenim receptorima.Cilj ovog diplomskog rada bilo je optimiranje uvjeta za određivanje signalnih molekula ERK i p38 MAPK iz THP-1 stanične linije Western blot metodom. Dobiveni rezultati pokazuju da su korištenjem protokola Cell Signaling Technology (CST) postignuti optimalni uvjeti i najbolji rezultati za sva četiri analizirana proteina: ERK, p-ERK, p38 i p-p38. Koncentracija proteina nanesenih na poliakrilamidni gel treba biti između 30 i 40 μg po jažici. Signal pozadine značajno je smanjen korištenjem nitrocelulozne membrane u odnosu na PVDF membranu. Vizualizacija proteina kemiluminiscencijom uz korištenje detekcijske otopine svježe pripremljene u laboratoriju daje konačnu sliku proteina koje se ne razlikuje od slike dobivene uz korištenje komercijalno dostupne otopine (Amersham ECL Prime; GE Healthcare Life Sciences).
Abstract (english)
Chronic obstructive pulmonary disease is characterized by a strong inflammatory response to inhalation of harmful irritating particles. Recruitment of immune cells that secrete cytokines, chemokines and proteases, consequently leads to airway obstruction and reduced air flow. TLR receptors are primarily responsible for innate immunity and they enhance the response of acquired immunity. There was a link between TLR receptors, activation (phosphorylation) of MAPK kinase (ERK, p38, JNK) and the secretion of various cytokines that cause an inflammatory response. TLR4 agonist (LPS) and TLR2 agonist (LTA) might be used for examination of the inflammatory response induced by these receptors. The aim of this study was the optimization of the conditions for determining the signaling molecules ERK and p38 MAPK from THP-1 cell lines by Western blot method. The results show that the optimal conditions and the best results for all the examined proteins ERK, p-ERK, p38 and p-p38 are achieved by using the protocol from Cell Signaling Technology (CST). The concentration of protein applied to the polyacrylamid gel should be between 30 and 40 μg per well. The background signal is significantly reduced by using a nitrocellulose membrane compared to PVDF membrane. Visualization of proteins with chemiluminescence detection solution freshly prepared in the laboratory, gives the final images of the proteins that do not differ significantly from the images obtained with a commercially available solution (Amersham ECL Prime; GE Healthcare Life Sciences).
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:163:993535
Study programmeTitle: Pharmacy
Study programme type: university
Study level: integrated undergraduate and graduate
Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
Type of resourceText
File originBorn digital
Access conditionsAccess restricted to students and staff of home institution
Terms of useLicence In copyright icon
Created on2016-10-27 09:58:58