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Since biological molecules are so important in human diagnosis, distinguishing them from other biological matrices has piqued interest in the natural sciences. In reality, distinguishing the chemical composition of healthy and sick people's proteins may provide valuable information about effective treatment options. For example, by studying the mechanism by which cancerous cells divide, it is possible to learn more about the specific protein that is changed during this process. These proteins, also known as biomarkers, are especially important in today's world, given the increasing number of cancer patients. Although existing research has concentrated on rare strains of cancer, it has failed to address the possibility of diagnosis for neuroendocrine tumors (NETs) before metastasis, and the low survival rates of concerned patients if proof. Presently, therefore, the scientific problem revolves around the observation that NETs can only be detected at the advanced stages. Accordingly, this scenario is problematic in that it renders the detection of cancerous development harder to observe. This problem is further compounded by the difficulty of separating low-abundant protein markers from complex mixtures of proteins. Resultantly, these challenges have presented the opportunity for possible solutions which apparently involve the optimization of one-dimensional polyacrylamide gel electrophoresis (ID PAGE). Through this optimization, protein compositions could be separated from samples of neuroendocrine tumors. In testing the above hypothesis, the methodology in the study involved lab experiments in which NET samples were collected from varied tumor locations and tested against control samples. Consequently, the findings of the study indicated that proteomic analyses could help isolate proteins that undergo alterations in tumor cells. The implication, therefore, is that by optimizing proteomic procedures, individual biomarkers could be identified for the earlier detection of cancer.
Notably, the preparation for the actualization of the SDS- Page technique also involved the preparation of cell cultures. Firstly, neuroendocrine tumors would be isolated in sterile conditions during surgery. Secondly, the tumor samples so obtained would then be cultured in RPMI-l640. The third step involved culturing in Petri dishes. However, the amount of connective tissues and fats would be reduced significantly before the sample was crushed, filtered through a variety of cell strainers before being centrifuged and subjected to osmotic shock. This treatment served the purpose of ensuring erythrocytes underwent hemolysis. Fourthly, the resultant pellet was resuspended before the counting and redistribution of individual cells. Lastly, supernatants were collected from the cultures after 72 hours, centrifuged, and distributed. After cell culture preparation, the next procedure involved the preparation of samples. During this stage, albumin was removed from supernatants and suspending medium. After that, protein samples were precipitated and concentrated. This process involved the use of acetone-methanol for the desalinization and concentration of samples. During testing, the SDS - Page technique involved the thawing and mixing of samples with a buffer comprising glycerol, Tris-HCl, β-mercaptoethanol, SDS and bromophenol blue. After mixing, the buffer would then be incubated and centrifuged. On the other hand, glycerol functioned to enhance the sample's density for application of SDS-capillary gel electrophoresis. Bromophenol blue acted as a staining agent to aid the observation of the process through which proteins separate. In the last stage, the analysis involved trypsin digestion of the bands of proteins, their alkylation, and further overnight digestion.
Miękus, Natalia, et al. "Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines." Molecular medicine reports 11.2 (2015): 1407-1415.
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