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Name of the research project (Highly specific and matching the aim of the project) Isolation of plasma membrane proteins by biological biotynilation via promiscuous BirA
Turkey (TurkMSIC) - Kocaeli University, Kocaeli
Department of Medical Biology,Kocaeli University Faculty of Medicine, Umuttepe Location 41380 Izmit/KOCAELI
Prof. Dr. Murat Kasap
Prof. Dr. Murat Kasap
Type of Research Project
- Basic science
What is the background of the project?
(Provide information about the project; min. 10 lines, max. 25 lines) Plasma membrane (PM) proteins participate in diverse biological processes including signal transduction, ion transport and membrane trafficking, and are the first responders in cell-environment communication. In a lot of researches, we need to isolate these proteins for identifying them. Enrichment of plasma membrane proteins and their subsequent identification is a challenging job (Yoneten, KK., Kasap M. et al., 2019). One of the methods used for enrichment is to biotinylate them via a chemical reaction and isolate them through streptavidin column. The efficacy of this method is low due to the nature of chemical biotinylation. In this project we will use an enzyme, BirA, to biotinylate plasma membrane proteins before we identify them with mass spectrometry. Kocaeli University Faculty of Medicine Department of Medical Biology has been established in 1995 and is working with 13 researchers.
What is the aim of the project?
Developing a new method for easier isolation of plasma membrane proteins via biological biotinylation and comparison of plasma membrane isolation speed between numerous methods.
What techniques and methods are used?
-Recombinant protein synthesis and purification: -Enzyme assay to measure either the disappearance of substrate over time or the appearance of product over time. -Plasma membrane protein enrichment -Cell culture: It is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. -Liquid chromatography–mass spectrometry (LC-MS): It is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyse biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin.
What is the role of the student?
- The student will mainly observe
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
What are the tasks expected to be accomplished by the student? (Minimum 10 lines, provide detailed explanation) The following tasks will be expected to be accomplished by the student: -Learning the usage of Statistical Package for the Social Sciences (SPSS) 26,statistical analysis, data management (case selection, file reshaping, creating derived data) and data documentation(a metadata dictionary is stored in the datafile) are features of the base software. -Analysis of proteins via mass spectrometry. - Learning cell culture procedure that involves the distribution of cells in an artificial environment (in vitro) which is composed of the necessary nutrients, ideal temperature, gases, pH and humidity to allow the cells to grow and proliferate -Observing to a researcher for learning principles of working in a lab (Hygiene rules, waste disposal rules etc.) and successfully applying during exchange.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Will there be any theoretical teaching provided for the student (preliminary readings, lectures, courses, seminars)? Please describe how much teaching and by whom. The following topics will be provided in the form of seminars by Prof. Dr. Murat Kasap. Lessons will be provided according to the availability of the teacher, at least once per week: -Cell culture protocols -Protein synthesis, translation and cell cycle - Liquid chromatography–mass spectrometry (LC-MS) Analyses Techniques -Cell structure and organelles -Enzyme and mechanisms
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
No specific skills are required.
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Students in biomedical fields
- Yoneten K K; Kasap M; Akpinar G; Kanli A; Karaoz E. Comparative Proteomics Analysis of Four Commonly Used Methods for Identification of Novel Plasma Membrane Proteins. J Membr Biol. 2019 Jul 25. doi:10.1007/s00232-019-00084-3. PubMed PMID: 31346646.
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