Computational protocol: Identification and Characterization of 5′ Untranslated Regions (5′UTRs) in Zymomonas mobilis as Regulatory Biological Parts

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Protocol publication

[…] Western blotting analysis was performed to detect GFP expression using anti-GFP antibody (Roche 11814460001). Standard western blotting protocols were used as previously described (Cho et al., ). Briefly, total cellular lysates were loaded onto a 12% denaturing SDS-PAGE gel. Gels were transferred to methanol activated PVDF membranes using the Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, CA) and run for 40 min at 15 V or 20 min at 25 V. Membranes were blocked with 5% dry non-fat milk in Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature. The proteins were detected with anti-GFP antibody at 1:1000 dilutions or anti-FLAG at 1:5000 (Proteintech, China). As a secondary antibody, goat anti-mouse IgG (H + L) HRP Conjugate (Promega #W4021) was used at a dilution of 1:2500 (or 1:5000 with Proteintech, China). All images were developed using Clarity™ ECL Western Blotting Substrate (BioRad, #170-5060) and the ChemiDocTM MP Imaging System (BioRad, CA) or West Dure Extended Duration Substrate Kit (AntGene, China) with AI600 Imaging System (GE, USA). Bradford assay measurements were used to normalize the loading of all protein analyses by total protein mass. Specific proteins were detected on the membrane by western blot analysis and quantified using ImageQuant TL 8.1 (GE Healthcare, CA) or Gel-Pro analyzer (LiuYi, China). Each protein was detected using anti-GFP. The level of GFP expression was measured and then normalized using the expression of RecA as an internal control. […]

Pipeline specifications

Software tools Imagequant TL, Gel-Pro Analyzer
Application DNA fingerprinting
Organisms Zymomonas mobilis
Chemicals Ethanol, Xylose