Journal: Journal of Cell Science
Article Title: Rapid production of pure recombinant actin isoforms in Pichia pastoris
Figure Lengend Snippet: Purification of recombinant human non-muscle actins and fission yeast Act1. (A) CBB gel image showing purified recombinant human β-actin (Actb), human γ-actin (Actg1) and Schizosaccharomyces pombe (SpAct1) actin. Immunoblotting in the bottom panels was performed with antibodies against Actb or Actg1 raised against peptides corresponding to the N-terminus of human β- and γ-actins. Since the N-terminus of S. pombe Act1 has strong similarity to human γ-actin, the anti-Actg1 antibody can recognize S. pombe Act1 as well as γ-actin (see sequence alignment shown at the bottom). (B) Actin sedimentation assays. Purified actin in low-ionic strength buffer (G-buffer) was mixed with KCl, EGTA and Mg 2+ to induce actin polymerization. Actin sedimentation was tested in the presence of 0.5 mM LatA or in solvent control (DMSO) and in the absence of DMSO or Latrunculin A (‘−’). After ultracentrifugation, actin in the pellet (Pellet) and supernatant (Sup) fractions were detected by CBB staining following SDS-PAGE. Rabbit muscle actin was used as a positive control. (C) Visualization of F-actin. The specified actins were incubated with KCl, EGTA and Mg 2+ in the presence of 2% PEG. F-actin in all cases was then stained with Rhodamine–phalloidin and imaged by using a Nikon inverted microscope fitted with a Yokogawa spinning-disc head and an Andor iXon camera. Scale bars: 5 µm.
Article Snippet: For imaging actin filaments, actin (8.33 µM) in G-buffer was polymerized by addition of 2 mM MgCl2 , 5 mM EGTA, 100 mM KCl and 2% polyethylene glycol 20,000 (#8.17018.1000, Merck Millipore) followed by incubation at 24°C for 1 h. 5% (v/v) Rhodamine–phalloidin (#R415, Thermo Fisher Scientific) or phalloidin–CF633 (#00046, Biotium) was added, and F-actin was imaged using an Andor Revolution XD spinning-disc confocal system on a Nikon Eclipse Ti inverted microscope, with a Nikon Plan Apo Lambda 100×1.45 NA oil immersion objective lens, a spinning-disc system (CSU-X1; Yokogawa) and an Andor iXon Ultra EMCCD camera.
Techniques: Purification, Recombinant, Sequencing, Sedimentation, Staining, SDS Page, Positive Control, Incubation, Inverted Microscopy