Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope.

Beyond the rewards of plant genome analysis and gene identification, characterisation of protein activities, post-translational modifications and protein complex composition remains a challenge for plant biologists. Ideally, methods should allow rapid isolation of proteins from plant material achieving a high degree of purity. We tested three purification strategies based on the eight-amino acid StrepII, six-amino acid His(6) and 181-amino acid Tandem Affinity Purification (TAP) affinity tags for enrichment of a membrane-anchored protein kinase, Nt CDPK2, and a soluble protein, At SGT1b, from leaf extracts.

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation.