Nts respectively), four runs for each operator are shown. Relative potencies

Nts respectively), four runs for each operator are shown. Relative potencies were similar for all the tests performed demonstrating consistent performance by two operators. doi:10.1371/journal.pone.0049516.g0?25 nM. Sensorgram curves were fitted to a 1:1 Langmuir binding model (BIAevaluation 3.0 software, GE Healthcare). The association, ka (1/Ms), the dissociation, kd (1/s) and the equilibrium, KD (KD = kd/ka) (M) constants were determined.BoNT/A treatmentCells were treated with BoNT/A complex, 150 kDa BoNT/A (Metabiologics, Madison, WI), recombinant LHN/A or iBoNT/A, or with BOTOXH (Allergan, Irvien, CA) for different amounts of time as specified in each assay. The medium containing BoNT/A was then replaced with fresh medium without toxin and cells were incubated for additional periods of time as specified in each assay. Each concentration of BoNT/A was tested in triplicate.Cell Lysis and Western Blot AnalysisCells 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 tabletof EDTA-free protease inhibitors) on ice for 20 min. Lysed cells were centrifuged in the plate at 4000 rpm for 20 min at 4uC. Western blots (WB) for detection of SNAP25206 and SNAP25197 or only SNAP25197 were described previously [51]. For WB analysis the lysates were transferred to a 96-well PCR plate, 26 SDSPAGE loading buffer (buy KS 176 Invitrogen) was added, and the plate was heated to 95uC for 5 minutes. The gels were run in 16 MOPSSDS 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 16 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 [51] diluted 1:1000, SMI-81 antibody (Sternberger Monoclonals Inc) diluted 1:5000 to evaluate the intact and cleaved SNAP25 products (i.e. SNAPSensitive Cell-Based Potency Assay for BoNT/Aand SNAP25197 were detected). Antibodies were diluted in 2 24272870 blocking agent/TBS-T buffer and incubated overnight at 4uC with gentle shaking. Secondary antibodies were MedChemExpress Tunicamycin anti-rabbit or antimouse 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 lex 452/ lem 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.SuperSignal ELISA Pico 1:1 mixture (Pierce) and read at 395 nm on a Luminometer (Molecular Devices). Data was fitted to a 4PL model as detailed above.Supporting InformationFigure S1 The ECL-ELISA CBPA with SiMa cells can be used to test the biological activ.Nts respectively), four runs for each operator are shown. Relative potencies were similar for all the tests performed demonstrating consistent performance by two operators. doi:10.1371/journal.pone.0049516.g0?25 nM. Sensorgram curves were fitted to a 1:1 Langmuir binding model (BIAevaluation 3.0 software, GE Healthcare). The association, ka (1/Ms), the dissociation, kd (1/s) and the equilibrium, KD (KD = kd/ka) (M) constants were determined.BoNT/A treatmentCells were treated with BoNT/A complex, 150 kDa BoNT/A (Metabiologics, Madison, WI), recombinant LHN/A or iBoNT/A, or with BOTOXH (Allergan, Irvien, CA) for different amounts of time as specified in each assay. The medium containing BoNT/A was then replaced with fresh medium without toxin and cells were incubated for additional periods of time as specified in each assay. Each concentration of BoNT/A was tested in triplicate.Cell Lysis and Western Blot AnalysisCells 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 tabletof EDTA-free protease inhibitors) on ice for 20 min. Lysed cells were centrifuged in the plate at 4000 rpm for 20 min at 4uC. Western blots (WB) for detection of SNAP25206 and SNAP25197 or only SNAP25197 were described previously [51]. For WB analysis the lysates were transferred to a 96-well PCR plate, 26 SDSPAGE loading buffer (Invitrogen) was added, and the plate was heated to 95uC for 5 minutes. The gels were run in 16 MOPSSDS 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 16 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 [51] diluted 1:1000, SMI-81 antibody (Sternberger Monoclonals Inc) diluted 1:5000 to evaluate the intact and cleaved SNAP25 products (i.e. SNAPSensitive Cell-Based Potency Assay for BoNT/Aand SNAP25197 were detected). Antibodies were diluted in 2 24272870 blocking agent/TBS-T buffer and incubated overnight at 4uC with gentle shaking. Secondary antibodies were anti-rabbit or antimouse 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 lex 452/ lem 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.SuperSignal ELISA Pico 1:1 mixture (Pierce) and read at 395 nm on a Luminometer (Molecular Devices). Data was fitted to a 4PL model as detailed above.Supporting InformationFigure S1 The ECL-ELISA CBPA with SiMa cells can be used to test the biological activ.