Purification and Characterisation of Proteins
Reinigung und Charakterisierung von Proteinen
8 CPSummerEN
Prerequisites
Mandatory, as written
Only applies to the Master Biotechnology PO 2018 with 4 semesters standard period of study: Successfull completion of module • 12739 - Research Internship
Contents
The experimental work of the students is integrated in actual research projects (proteolytic systems, proteasome regulators, apoptosis, protein modification etc). Basic techniques such as homogenization, centrifugation and salt precipitation, as well as ion exchange chromatography and other FPLC techniques are applied to research topics of the biochemistry unit. In a second step the composition of purified protein fractions is analyzed simply by SDS PAGE and Western blotting or via isoelectric focusing (IEF) and non-equilibrium pH gradient gel electrophoresis (NEPHGE). Enzyme assays and immune-detection methods are applied to monitor the quality of target proteins during protein fractionation. Fractionated salt precipitation, ultracentrifugation and ion exchange chromatography are the basic methods to purify proteasome regulators and their functional mutants. Other proteins may be useful for the standardization of immune-detections.
Learning outcomes
Lab Course The course is aimed to train master students on basic techniques in protein biochemistry and proteome research. To develop strategies for the purification and characterization of various proteins is the major goal of this course. Another didactic aim is to consolidate problem solving abilities and structured experimental team work as well as individual responsibilities. During a lab course students are working individually or as a team depending on the complexity of the analytic task to strengthen team work, as well as individual responsibility. Technical competences in protein purification are reaching from culturing prokaryotic and eukaryotic cells, providing cell lysates, via enrichment of proteins by subcellular fractionation towards classical strategies of salt precipitation, ultrafiltration and chromatography. Characterization encompasses electrophoreses (NEPHGE, IEF, SDSPAGE) and immune-identification (immunoblotting). Functionality of proteins is accessed by assays for enzymatic activites or protein-protein interaction depending on the specific research task provided. Seminar Since each student experiences various purification approaches associated with specific problems, the weekly research seminar provides the opportunity to makes this knowledge available for each participant of the lab course. During oral presentations the participants introduce into relevant literature, covering the theoretical background and principles of methods applied. Scientific questions, experimental approaches and results of individual projects are presented as well. These compulsory seminars are strengthening the student´s conceptual skills by planning their experimental activities for the next week, by drawing conclusions from experimental data. Certainly, the seminars are also aimed to promote the development of professional communication skills.
Assessment
• Practical laboratory work 50% • Presentation and discussion in the seminar, duration depends on the topic (25%) • Lab work protocol, 10-20 pages (25%)
Literature (11)
- Practical Approach Series (David Hames, Editor; 185 volumes since 1981; IRL Press; Oxford University Press). Topics:
- Animal Cell Culture (3rd Ed.: Masters, R.W., 2000);
- Protein Function (2nd Ed.: Creighton, T. E. 1997);
- Protein Purification Techniques (2nd Ed.: Roe, S.2001);
- Subcellular Fractionation (Graham, J. & Rickwood, D. 1997);
- Proteolytic Enzymes (Beyon, R., 2001)
- Coligan, Dunn, Speicher, Wingfield (2002) Short Protocols in Protein Science, Wiley
- Barker, Kathy (2005). At the Bench: A Laboratory Navigator ISBN-13: 978-0879697082
- Original publications (depending on the assigned project).
- Protocols and manuals of the manufacturers of reagents and kits used during the lab course are provided as pdf-files using the university intranet. Seminar literature will be provided upon request.
- To strengthen professional skills, students have do develop their information acquisition strategies. Hence, seminar/lab course participants have to supplement provided information/material by their independent contributions.
Remarks
The module takes place as a 6 week block course.
Source: BTU's module description · retrieved 28 Sep 2026Original