Purification of the Ndc80 kinetochore subcomplex from Xenopus eggs.
The identification of protein binding partners often facilitates understanding of protein complex function. However, identifying binding partners has proven difficult because proteins are often bound to insoluble structures or are only present during certain stages of the cell cycle. Fortunately, Xenopus eggs stockpile many proteins, which are typically insoluble, as soluble subcomplexes to facilitate rapid early embryonic divisions. We exploited this by developing a purification scheme using Xenopus egg extracts to isolate Coomassie-stainable amounts of the xNdc80 kinetochore complex. In this scheme Xenopus eggs are directly made into a mitotic high-speed supernatant and then flowed over three chromatographic columns: heparin, Mono-Q, and Superose 6 gel filtration columns. A final immunoprecipitation is then performed from the peak Superose 6 column to yield Ndc80 complex purified to homogeneity. With minor modification and manipulation of Xenopus egg extracts, this protocol can easily be adapted for purification of other protein complexes.
McCleland
Mark L
ML
Department of Biochemistry and Molecular Genetics, University of Virginia Medical School, Charlottesville, VA, USA.
Stukenberg
P Todd
PT
D000818:Animals; D000837:Anion Exchange Resins; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents; D005260:Female; D006493:Heparin; D047468:Immunoprecipitation; D018386:Kinetochores / Q000737:chemistry*; D008938:Mitosis; D009687:Nuclear Proteins / Q000302:isolation & purification*; D010063:Ovum / Q000737:chemistry* / Q000166:cytology; D012117:Resins, Synthetic; D029867:Xenopus Proteins / Q000302:isolation & purification*; D014982:Xenopus laevis
D000837:Anion Exchange Resins; D004396:Coloring Agents; D009687:Nuclear Proteins; D012117:Resins, Synthetic; D029867:Xenopus Proteins; C042855:Mono Q; D006493:Heparin