Biochemical Methods
In order to analyze an interaction of proteins of interest, the first thing to do is to produce and purify it. Using the proper expression vectors the respective protein will be synthesized in a bacterial culture and purified in two or three steps with different chromatographic methods. Standard techniques of molecular biology (in particular PCR, polymerase chain reaction) allow to construct the desired types of protein. First of all insertion of particular point mutation or deletion of one or more protein domains, followed by biophysical analysis of a modified properties and comparison with a wildtype permits to a significant interference on the functionality of the protein.
Protein purification
Cultivation of E.coli
Cultivation of bacterias (E.coli) in a fermenter or in Erlenmeyer flasks in a shaking incubator. At the right cell density adding of a chemical will induce the bacterial synthesis of the desired protein
Cell harvesting
Centrifuges of different sizes allow the harvest of microbiological culture and separation of insoluble components after cell lysis.
Cell Lysis
Lysis of bacteria (breaking of cell walls) by supersonic sounding.
Äkta Purifier
Chromatography anlage with different type of column for purification of recombinant protein from Bacterial lysat. The picture shows the Akta Purifier-System
SDS-PAGE
The purity of isolated protein will be verified by electrophoresis. This picture shows analysis of a chemical cross-link kinetics on a SDS-polyacrylamid-Gel. During the reaction, the MST1-Kinase fragments are linked as tetramers, while dimer and trimer occur as intermediate products
UV/Vis- Spektroscopy
The essential requirements of a quantitative analysis of the protein interaction is exact definition of the concentration of involved interaction member. Using the UV/Vis- Spektroscopy (picture) we can utilize different methods (Gill&von Hippel, Bradford etc.)