Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.bastieen

Authors: Arindam Naha

Summary: For studying intracellular accumulation of CTB subunits in E coli, bacteria were grown up to 4 hours following induction by 0.2% of arabinose. Post induction, bacterial cultures were mixed with equal volume of translation-translocation halt cocktail (200 µg/ml chloramphenicol, 200 mM sodium azide, 9.5% ethanol) and immediately placed in an ice water bath for 15 minutes of incubation to arrest protein synthesis and translocation. Cells were harvested by centrifugation at 14000 rpm for 15 minutes at 4°C. Supernatants were preserved and used for CT-ELISA experiment as described before. Pellets were then suspended in TME buffer (20 mM Tris-Cl pH 8.0, 2mM β-mercaptoethanol, 1 mM EDTA), preferably 1/10th volume of the original culture and disrupted by sonication. After centrifugation at 13,000 rpm at 4°C for 15 min to remove any unbroken cells, crude cell lysates were used for measurement of protein concentration using Bradford assay (BioRad, USA). Equal amount (≥80 μg) of protein samples were mounted on to 15% SDS-PAGE. Blots were probed with classical CTB-specific monoclonal antibody (anti-Cla CTB, 1:5000) or anti-beta lactamase (anti-βla 1:500) antibody. A portion of the same blot was stained with Ponceau S staining solution (Sigma-Aldrich, St Louis, MO, USA) to validate whether equal amount protein samples were transferred in the blot. Blots were developed using West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions and visualized in ChemiDoc XRS+ system (BioRad, USA). Densitometric analysis of band intensities was performed using the Multi Gauge software V2.3(FujiFilm). .justify:after { content: ""; display:inline-block; width: 100%; }

Associated Publications: Naha A, Mandal RS, Samanta P, Saha RN, Shaw S, Ghosh A, Chatterjee NS, Dutta P, Okamoto K, Dutta S, Mukhopadhyay AK (2020) Deciphering the possible role of ctxB7 allele on higher production of cholera toxin by Haitian variant Vibrio cholerae O1. PLoS Negl Trop Dis 14(4): e0008128. doi: 10.1371/journal.pntd.0008128

Affiliations: Post Doctoral Fellow, Indian Institute of Science (Previously Ph.D Student. National Institute of Cholera and Enteric Diseases)

External URL: https://doi.org/10.1371/journal.pntd.0008128

Version: 1

Publication Date: 2019

Expand All Collapse