Joyce Mulder
  • Graduate, Health, NIZO, The Netherlands, The Netherlands,
Research fields
  • Microbiology
Purification and TEM of Afp and Its Variants
Authors:  Daria Rybakova, Alok K. Mitra and Mark R. H. Hurst, date: 05/20/2014, view: 9771, Q&A: 0
The Serratia entomophila antifeeding prophage (Afp), forms a phage-tail-like particle (tailocin) that causes cessation of feeding activity of the New Zealand grass grub, Costelytra zealandica. Here, we describe the more detailed purification protocol for Afp particles and its variants which is based on the procedure described in our original publication (Rybakova et al., 2013). The purification procedure includes inducing Escherichia coli (E. coli) cells harbouring afp genes under arabinose-inducible promoter for 24 h. The cells are harvested and sonicated on ice followed by DNAse treatment and centrifugation. The supernatant is then filter sterilised and applied to the size exclusion chromotography (SEC) column. The fractions containing Afp or its variants are pooled and ultracentrifuged. The supernatant is removed and the transparent pellet is resuspended in the residual buffer. The procedure results in Afp particles or variants thereof that are approximately 70% pure. The Afp particles are negatively stained and visualized using Transmission electron microscopy (TEM).
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