Izoelektrično fokusiranje proteina stanične stijenke kvasca Saccharomyces cerevisiae (CROSBI ID 360163)
Ocjenski rad | diplomski rad
Podaci o odgovornosti
Gradvol, Vedran
Strelec, Ivica
hrvatski
Izoelektrično fokusiranje proteina stanične stijenke kvasca Saccharomyces cerevisiae
U ovom radu su nekovalentno i kovalentno vezani proteini stanicne stijenke razlicitih mutanata kvasca koji su se razlikovali u proteinima stanicne stijenke ekstrahirani pomocu 20 mM NaOH i 20 IU/μL beta-1, 3-glukanaze, nakon cega je uslijedilo izoelektricno fokusiranje u gradijentu pH 3-10, te nespecificna i specificna detekcija proteina. Nespecificna detekcija proteina je provedena bojanjem srebrom, a specificna detekcija imunokemijski nakon kapilarno-difuzijskog prijenosa proteina na PVDF membranu. Nespecificnom detekcijom proteina htjelo se ustanoviti da li postoje razlike u kolicini ekstrahiranih proteina i rasporedu proteinskih vrpci razlicitih mutanata kvasca, a imunokemijskom detekcijom se htjelo utvrditi prisustvo nekovalentno vezanih proteina Scw4p, Scw10p, Scw11p i Bgl2p oznacenih hemaglutininskim epitopom. Iz provedenog izoelektricnog fokusiranja, odnosno nakon nespecificne detekcije bojanjem srebrom mogu se uociti razlike u ekspresiji proteina pojedinih mutanata kvasca u vidu razlika u intenzitetu pojedinih proteinskih vrpci, dok istovremeno nema evidentnih razlika u samom rasporedu proteinskih vrpci. Prilikom razdjeljivanja proteina došlo je i do taloženja proteina na mjestu nanošenja što se otklonilo dodatkom Tritona X-100 u uzorke ekstrahiranih proteina. Imunokemijskom detekcijom pokazano je nespecificno vezanje proteina što je vjerojatno uzrokovano pucanjem stanica kvasca prilikom ekstrakcije i nespecificne reakcije unutarstanicnih proteina sa HAprotutijelima.
Saccharomyces cerevisiae; stanicna stijenka; kovalentno vezani proteini; nekovalentno vezani proteini; izoelektricno fokusiranje; imunokemijska detekcija
nije evidentirano
engleski
Isoelectric focusing of Saccharomyces cerevisiae cell wall proteins
Covalently and noncovalently linked cell wall proteins from different yeast protein mutants were extracted with 20 mM NaOH and 20 IU/μL -1,3-glucanase. Extracted proteins were separated by isoelectric focusing in a pH gradient of 3-10, and subsequently nonspecific and specific detection was carried out. Nonspecific detection was achieved by silver staining and specific detection was achieved immunochemically after the transfer of proteins to a PVDF membrane. The goal of nonspecific detection was to determine the differences in the quantity of extracted proteins and/or position of protein bands for the different yeast mutants, and the goal of specific detection was to determine the presence of noncovalently linked proteins Scw4p, Scw10p, Scw11p and Bgl2p tagged by haemaglutinin. Silver stained gels showed differences in protein expression of different yeast mutants that can be seen as differences of protein band intensities, but there were no evident differences in protein band positions of different yeast mutants. Protein precipitation during sample entry was omitted by addition of Triton X-100 in all samples of extracted proteins that increased protein solubility. Immunochemical detection showed nonspecific reaction of HA-antibodies with yeast proteins which was probably caused by the release of intercellular proteins after cell breakage during extraction.
Saccharomyces cerevisiae; cell wall; covalently linked proteins; noncovalently linked proteins; isoelectric focusing; immunochemical detection
nije evidentirano
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30.09.2009.
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