Recombinant T-Rp3 Lab Report

MATERIAL AND METHODS
Recombinant protein production
The recombinant T-Rp3 was expressed in E. coli BL21 (DE3) as previously described (Favaro et al., 2014) and purified by a single step His-based affinity chromatography in an ÄKTA purifier FPLC (GE Healthcare). Protein was then dialyzed against 50 mM MES buffer (pH 6,0) overnight at room temperature, frozen in liquid nitrogen and stored at -80 ˚C after 0.22 μm pore membrane filtration.

Evaluation of DNA-protein interaction by gel retardation assay
Protein-DNA complexes were formed using 1 µg of plasmid pTriEx-1.1 Hygro at different pDNA: protein molar ratios (1:100, 1:200, 1:500, 1:1000, 1:2000, 1:8000) during 10 minutes. The increasing molar ratios were assessed by gel retardation assay on a 0.8% agarose gel and visualized by ethidium bromide staining. For further experiments, all samples were prepared in the 1:8000 pDNA:protein molar ratio with 1
Samples were submitted to negative staining with uranyl acetate and observed with a Jeol JEM 1400 transmission electron microscope, equipped with a CCD Gatan ES1000W Erlangshen
Control experiments were performed in the same conditions but without the addition of chloroquine. The medium was replaced 6 hours post-transfection and cell samples were analyzed 24 hours post-transfection after treatment with 1 mg/mL trypsin (Gibco) for 15 minutes on a FACS¬Canto system (Becton Dickinson) using a 15 mW air-cooled argon ion laser at 488 nm excitation. Fluorescence emission was measured with a D detector (530/30 nm band pass