Title Karakterizacija novoproizvedenog protutijela protiv CD63 proteina u sisavaca Title (english) Characterization of newly produced antibody against CD63 protein in mammals Author Roberta Padavić Mentor Kristina Grabušić (mentor) Mentor Mladenka Malenica Staver (komentor) Committee member Elitza Petkova Markova-Car (predsjednik povjerenstva) Committee member Kristina Grabušić (član povjerenstva) Committee member Mladenka Malenica Staver (član povjerenstva) Committee member Natalia Kučić (član povjerenstva) Granter University of Rijeka Defense date and country 2020-06-03, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract CD63 je membranski glikoprotein koji pripada superobitelji tetraspanina. Molekularna masa CD63 iznosi 25 kDa, ali može doseći i do 60 kDa zbog različitih stupnjeva glikozilacije ovog proteina. Prisutan je u svim ljudskim stanicama, te ima mnogo potvrđenih i još više ne potvrđenih funkcija. CD63 se u stanici nalazi u endosomalnom sustavu, egzosomima i na površini stanice. Uključen je u signalnu transdukciju koja regulira razvitak, aktivaciju, rast i pokretljivost stanice. Promovira preživljenje stanice, reorganizaciju citoskeleta, adheziju, migraciju, te je uključen u transport proteina. Jako je eksprimiran u ranim fazama raznih tumora, a u kasnijim fazama ga gotovo uopće nema. Ima ulogu i u nekim drugim patološkim stanjima, te bi mogao imati izvanrednu dijagnostičku, prognostičku i terapijsku vrijednost i služiti kao marker za stadije tumora.
Cilj ovog rada je optimizacija imunofluorescencije i karakterizacija novoproizvedenog anti-CD63 protutijela (hCD63.01).
Provedeno je više pokusa imunofluorescencije na HeLa, SW620 i MCF-7 staničnim linijama. Mijenjane su koncentracije sredstva za permeabilizaciju i sredstva za blokiranje, te i samo sredstvo za permeabilizaciju. Za usporedbu je korišteno MEM-259 anti-CD63 protutijelo. Kako bi odredili je li novoproizvedeno protutijelo prošlo kroz promjene tijekom pročišćavanja, provedena je imunofluorescencija sa nepročišćenim supernatantom hCD63.01, te su rezultati uspoređeni sa onima pročišćenog protutijela hCD63.01.
Utvrđeno je da hCD63.01 protutijelo nije značajno promijenjeno tijekom pročišćavanja. Predložen je optimizirani protokol imunofluorescencije sa Tween-om 20 kao sredstvom permeabilizacije. hCD63.01 je u svim pokusima dalo jači signal od MEM-259 komercijalno dostupnog protutijela za CD63., te je moguće da hCD63.01 prepoznaje više formi ovog proteina. Dodatna istraživanja su potrebna za potpunu karakterizaciju i razumijevanje potencijala hCD63.01 novoproizvedenog protutijela.
Abstract (english) CD63 is a membrane glycoprotein which belongs to a superfamily of tetraspanins. Its molecular weight is 25 kDa but can reach up to 60 kDa depending on the level of glycosylation the protein went through. It is present in all human cell types and has lots of different functions some of which are known and some of which have yet to be proven. CD63 can be found in the cell endosomal system, egzosomes and on the cell surface. It is involved in signal transduction which regulates cell development, activation, growth and motility. It promotes cell survival, reorganization of actin cytoskeleton, adhesion, cell migration and is included in protein transport. It is highly expressed in early stages but almost nonexistent in the later stages of most tumors and has a role in some other pathological states. CD63 could have an excellent diagnostic, prognostic and therapeutical value for different pathological conditions and be used as a stage marker for various tumors.
Aim of this paper is the optimization of immunofluorescence and characterization of a novel anti-CD63 antibody (hCD63.01).
Several immunofluorescence experiments were conducted on HeLa, SW620 and MCF-7 cell lines. Changes in the protocol were made regarding concentration of the permeabilization reagent and the blocking buffer. Different permeabilization reagents were used. Apart from the hCD63.01 antibody, a commercial anti-CD63 antibody (MEM-259)was used for comparison. Supernatant of nonpurified hCD63.01 was also used in order to establish possible changes which may have occurred in the antibody purification process.
It was established that hCD63.01 did not went through significant changes during purification. Optimized immunofluorescence protocol was proposed with Tween 20 as the permeabilization reagent. In every experiment hCD63.01 had a stronger signal than MEM-259 commercially available CD63 antibody. It is possible that hCD63.01 recognizes more forms of CD63. Further studies are needed for the complete characterization and understanding of hCD63.01 antibody’s potential.
Keywords
CD63
imunofluorescencija
protutijelo
hCD63.01
Keywords (english)
CD63
immunofluorescence
antibody
hCD63.01
Language croatian URN:NBN urn:nbn:hr:193:012435 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Repository Repository of the University of Rijeka, Faculty of Biotechnology and Drug Development Created on 2020-07-02 09:07:10
