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Protein Purification2018-11-21T11:48:42+00:00

Proteins can be extracted from lysed cells or from culture supernatant, and are purified using all common purification methods and analysed using appropriate methods for purity and identity. We have a wealth of published work describing the use of protein purification and analytical approaches to support drug discovery

Cells can be lysed at scale under high pressure homogenisation, using sonication or more gently by freeze thaw as appropriate. Alternatively, secreted proteins are purified from cell culture media. Both eukaryotic systems use protein free media, greatly simplifying the purification process. All the standard affinity chromatographic procedures are used regularly with a variety of different tags. In addition proteins are purified according to size, charge, hydrophobicity and other properties depending on the protein characteristics. A suite of Akta systems are available for the work.

Proteins are analysed by a variety of methods both during method development and as a final QC. SDS-PAGE is used under both reducing and non-reducing conditions as required, to look for disulphide bonds. Purity and size in solution is determined using size exclusion chromatography.

Protein concentration is determined using absorbance at 280nm, correcting for the extinction coefficient. LC mass spectrometry is used in two ways:  to determine the intact mass of the protein to search for post-translational modifications if feasible and to peptide map after proteolytic digestion. The thermal stability of the protein can also be monitored using a thermal shift assay. This can be particularly useful to infer a folded state and to monitor compound binding if this shifts the melting point.

Post-translational modifications are very important to the folding and function of many proteins. We have significant experience of analysing these modifications using mass spectrometry and altering them as required. We have worked with proteins that are N and O-glycosylated, disulphide bonded, phosphorylated, lipid modified, proteolytically activated, poly-ADP ribosylated and many other modifications.

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