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BoNT/E-directed Western blot

MN Maria B. Nowakowska
KS Katja Selby
AP Adina Przykopanski
MK Maren Krüger
NK Nadja Krez
BD Brigitte G. Dorner
MD Martin B. Dorner
RJ Rongsheng Jin
NM Nigel P. Minton
AR Andreas Rummel
ML Miia Lindström
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Aliquots of C. botulinum Beluga WT and Beluga Ei culture S/Ns before and after trypsinization were separated in 4–12% SDS-PAGE. Purified 15 ng BoNT/E standard was used as a Western blot reference. Proteins were transferred onto a PVDF 0.2 µM membrane, subsequently blocked and incubated in BoNT/E-directed KE97 rabbit polyclonal antibodies (1:1,250 in PBS with 0.05% Tween 20 (PBST) with 5% bovine serum albumin) at 4 °C overnight. The membrane was washed, incubated in goat anti-rabbit IgG:HRP antibody (Bio-Rad, Hercules, CA, USA) (1:50,000 in PBST) for 2 h at RT and washed again. Subsequently, the membrane was incubated in Clarity Western solution (Bio-Rad) and chemiluminescence was detected using Fujifilm LAS-3000 Imager (Fuji Photo Film, Tokyo, Japan) applying different exposure times.

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