Application of different methods in determining protein concentration in a biological sample

Proteini su molekule bitne za strukturu, funkciju i regulaciju gotovo svih živih organizama. To su makromolekule sastavljene od aminokiselina koje su povezane peptidnim vezama u polipeptidne lance koji se savijaju u specifičnu trodimenzionalnu strukturu kako bi stvorili funkcionalne proteine. Određivanje koncentracije proteina u biološkim uzorcima ključno je za razumijevanje bioloških procesa, u dijagnostičke svrhe te za terapiju raznih bolesti. Koncentracija određenih proteina van referentnih vrijednosti može biti povezana sa specifičnim bolestima ili stanjima. Kvantifikacija ukupnih proteina u uzorku često je prvi korak u kompleksnijim analizama koje zatim služe boljem razumijevanju bioloških funkcija proteina i samim time boljem liječenju. Cilj ovog istraživanja bio je utječe li primjena različitih analitičkih metoda na vrijednost koncentracije proteina u biološkim uzorcima tkiva i seruma. Koncentracija ukupnih proteina određena je u uzorcima mozga, debeloga crijeva i seruma miša soja C57BL/6 pomoću 4 metode: metode bicinkoninskom kiselinom (engl, bicinchoninic acid assay, BCA), bromfenol plavo, Bradford i biuret metode. Uslijed interakcije neke od komponenti korištenog reagensa i proteina iz uzorka kod svih navedenih metoda, dolazi do promjene apsorpcije svjetlosti u uzorcima koja se potom mjeri pomoću spektrofotometra. Statistička analiza pokazala je kako se rezultati dobiveni Bradford metodom razlikuju od ostale tri metode u sve tri vrste uzorka, dok između BCA, bromfenol plavo i buiret metode nema statistički značajne razlike niti u jednom uzorku. Iz dobivenih rezultata može se zaključiti da je za određivanje koncentracije proteina u mišjem serumu, mozgu i crijevu prihvatljivo koristiti BCA, bromfenol plavo ili biuret metodu. Odabir metode ovisit će o cilju istraživanja, osjetljivosti, vrsti i količini uzorka te raspoloživim resursima. Iz ovog se istraživanja može zaključiti da kombinacija više metoda osigurava pouzdaniju karakterizaciju proteina u biološkim uzorcima. Također je bitno paziti na pripremu, starost i održavanje kemikalija i uvjete pohrane bioloških uzorka kako ne bi došlo do neispravnih rezultata.Proteins are molecules essential for the structure, function and regulation of almost all living organisms. These macromolecules are composed of amino acids connected by peptide bonds to form polypeptide chains that bend into a specific three-dimensional structure to create functional proteins. Determination of protein concentration in biological samples is crucial for understanding biological processes, for diagnostic purposes, and for the therapy of various diseases. The concentration of certain proteins outside the reference values may be associated with certain diseases or conditions. Quantification of total proteins in a sample is often the first step toward more complex analyses that then serve to better understand the biological functions of proteins and thus enable better treatment. The aim of this study was to determine whether the useof different analytical methods will affect the obtained value of protein concentration in biological tissue and serum samples. The concentration of total proteins was determined in brain, colon, and serum samples of C57BL/6 mice using four different approaches: bicinchoninic acid assay (BCA), bromphenol blue, Bradford and biuret methods. All of the aforementioned methods rely on the change in light absorption in the samples due to the interaction between the components of the reagent used and the protein of the sample. The light apsorption change is then measured spectrophotometrically. Statistical analysis showed that the results obtained by the Bradford method differed from those obtained by the other three methods for all three sample types, while there were no statistically significant differences between the BCA, the bromophenol blue, and the buiret method for any of the analyzed samples. According to our results, it can be concluded that the use of the BCA, bromophenol blue or buiret method for the determination of protein concentration in mouse serum, brain and intestine is an acceptable approach. The choice of the method used depends on the research objective, sensitivity, type and amount of the sample, as well as available resources. In evaluating the results of this study, it can be deduced that the combination of multiple methods provides more reliable characterization of proteins in biological samples. It is also important to pay attention to the freshness and maintenance of the prepared chemicals, as well as storage conditions of the biological samples to avoid false results