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Prof. Dr. Diana Imhof

University Bonn

Pharmaceutical Institute

Pharmaceutical Biochemistry and Bioanalytics

An der Immenburg 4

D-53121 Bonn

 

Telefon: 0228 73 5254
Fax: 0228 73 6829
E-Mail: dimhof [at] uni-bonn.de

 

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You are here: Home Areas AK Imhof Core Facility Services and devices Protein synthesis and purification

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.
S1 facilities


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



Aekta
Äkta Prime plus (GE Healthcare)
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.



MSMS
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.
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