216461b
Laboratory Course
WiSe 22/23: Production and Biophysical Analysis of Selected Membrane Proteins Part 1
Ramona Schlesinger
Information for students
Attention: The seminar and practical must be attended together with Prof. Heberles' course 216462
The open positions are given to the same students for both courses
Interested students, without official placement, can have their names put on a waiting list (write to r.schlesinger@fu-berlin.de) close
The open positions are given to the same students for both courses
Interested students, without official placement, can have their names put on a waiting list (write to r.schlesinger@fu-berlin.de) close
Comments
Content of the practical:
- Expression of two membrane proteins in E. coli and P. pastoris system
- purification via affinity chromatography (includes cell disruption with pressure, solubilization procedures with detergents) and gelfiltration on an Äkta system (demo)
- protein analysis with UV/Vis spectroscopy and SDS-PAGE
- sequential centrifugation for membrane protein purification
- Directed mutagenesis by PCR (megaprimer method, QuikChange) and cloning into the expression host P. pastoris (contains techniques like digestion and ligation of DNA, DNA prep, electrotransformation, agarose gel electrophoresis)
- Membrane protein crystallization trials in lipidic cubic phase
- the generated membrane proteins will be used in the subsequent course 216462 of AG Heberle