Computational protocol: Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

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Protocol publication

[…] Cells were washed once with PBS and lysed in freshly prepared Triton X-100 Lysis Buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, and one tablet of EDTA-free protease inhibitors) on ice for 20 min. Lysed cells were centrifuged in the plate at 4000 rpm for 20 min at 4°C. Western blots (WB) for detection of SNAP25206 and SNAP25197 or only SNAP25197 were described previously . For WB analysis the lysates were transferred to a 96-well PCR plate, 2× SDS-PAGE loading buffer (Invitrogen) was added, and the plate was heated to 95°C for 5 minutes. The gels were run in 1× MOPS-SDS running buffer (Invitrogen) at 200 V for 55 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad) pre-wet in Western blot transfer buffer (Invitrogen) containing 20% Methanol (Burdick and Jackson). The Western blot transferred at 800 mA for 2 h using the TE-62 transfer cell apparatus (GE Healthcare). Blots were blocked in 2% ECL blocking agent (GE Healthcare) in 1× TBS/0.1% Tween 20 (Bio-Rad) (TBS-T) for 1 h at room temperature. The following primary antibodies were used: anti-SNAP25197 polyclonal antibody diluted 1∶1000, SMI-81 antibody (Sternberger Monoclonals Inc) diluted 1∶5000 to evaluate the intact and cleaved SNAP25 products (i.e. SNAP25206 and SNAP25197 were detected). Antibodies were diluted in 2% blocking agent/TBS-T buffer and incubated overnight at 4°C with gentle shaking. Secondary antibodies were anti-rabbit or anti-mouse IgG H+L HRP conjugated (Invitrogen) diluted 1∶5,000 or 1∶10,000 in 2% blocking agent/TBS-T buffer for 1 h at room temperature. The membranes were washed and exposed for 5 min to ECL Plus Western Blotting System Detection Reagents (GE Healthcare). Chemifluorescence was captured by scanning the blots in the Typhoon 9410 Imager (GE Healthcare) at λex 452/λem 520. The intensity of the gel bands were calculated using ImageQuant TL software (GE Healthcare). The data was analyzed using SigmaPlot v 10.0 (Systat Software Inc.) or PLA2.0 (Stegmann Systems). Intensity values were plotted against concentration of BoNT/A in log scale and fitted to a 4-parameter logistics function (Y = Y0+a/[1+(X/X0)b]) without constraints. Based on the fitted curves the EC50 values, corresponding to “X0”, were determined. […]

Pipeline specifications

Software tools Imagequant TL, SigmaPlot
Application Miscellaneous
Organisms Mus musculus, Homo sapiens
Diseases Neuroblastoma, Genetic Diseases, Inborn