Protein synthesis and purification

Standard molecular biology techniques are routinely applied in the core facility. This involves transformation of bacterial cells (e.g. E.coli) and recombinant protein expression.

The resulting protein is subjected through a series of steps to achieve the final quality and quantity. The workflow involved from a technical perspective is as follows:

Protein purification
Protein identity determination
Protein purity determination
Protein quantitation

Please find below details of the individual steps.

Furthermore, semi-synthetic methods such as native chemical ligation of peptide fragments is also done as a part of the methods offered.

Core Facility homepage

Peptide synthesis and purification

Bioanalytical methods for the characterization of peptides and proteins

Protein purification procedures such as the ones involving His-tagged proteins are carried out by size exclusion chromatography. The Äkta Prime plus device (GE Healthcare) and a chromatography fridge are put to use in this regard.

Protein identity is established by tryptic digest followed by ESI-MS/MS experiments. The core facility uses the LC-ESI micrOTOF-Q III (Bruker Daltonics GmbH) mass spectrometer.

Äkta Prime plus (GE Healthcare)

LC-ESI micrOTOF-Q III (Bruker Daltonics GmbH)

Protein purity is determined using SDS-PAGE gel electrophoresis experiments.

Protein quantity is determined by standard protein determination methods and in special cases (individual consultation required) by amino acid analysis.