Novex hrp chromogenic substrate tmb

Control PBMCs, with no agents applied, as well as the cell samples exposed to DMSO (vehicle control) and the tested agents were lysed (4 °C, 1 h) in a buffer containing 10 mM Tris-HCl (pH agent-7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl 2 , 0.1 M DTT, and protease inhibitors as described previously. 62, 63 Protein content was estimated according to the Lowry method 64 and the cell lysates were prepared for subsequent Western blotting analysis.
Protein samples (50 lg/lane) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 8.0 or 12.5% slab gels 65 and electrotransfered onto Immobilon P. 66 Membrane staining with 0.5% Ponceau S solution was performed to confirm equal protein loading and completeness of the transfer. The membranes were saturated in 5.0% skim milk in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 1 h at ambient temperature, and then incubated overnight with antibodies specific to Mcl-1 (1:1000), Bax (1:1000), PARP-1 (1:1000), and lamin B (1:1000), all from Santa Cruz Biotechnology Inc. Antigen recognition was performed with anti-rabbit IgG (whole molecule)-horse peroxidase or anti-goat IgG-horse peroxidase antibody produced in goat or rabbit, respectively. The antigen-antibodies complexes were detected with Novex HRP Chromogenic substrate (TMB) from Invitrogen or, alternatively, by chemiluminescence.

Control PBMCs, with no agents applied, as well as the cell samples exposed to DMSO (vehicle control) and the tested agents were lysed (4 °C, 1 h) in a buffer containing 10 mM Tris-HCl (pH agent-7.5), 300 mM NaCl, 1% Triton X-100, 2 mM MgCl 2 , 0.1 M DTT, and protease inhibitors as described previously. 62, 63 Protein content was estimated according to the Lowry method 64 and the cell lysates were prepared for subsequent Western blotting analysis.
Protein samples (50 lg/lane) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 8.0 or 12.5% slab gels 65 and electrotransfered onto Immobilon P. 66 Membrane staining with 0.5% Ponceau S solution was performed to confirm equal protein loading and completeness of the transfer. The membranes were saturated in 5.0% skim milk in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 1 h at ambient temperature, and then incubated overnight with antibodies specific to Mcl-1 (1:1000), Bax (1:1000), PARP-1 (1:1000), and lamin B (1:1000), all from Santa Cruz Biotechnology Inc. Antigen recognition was performed with anti-rabbit IgG (whole molecule)-horse peroxidase or anti-goat IgG-horse peroxidase antibody produced in goat or rabbit, respectively. The antigen-antibodies complexes were detected with Novex HRP Chromogenic substrate (TMB) from Invitrogen or, alternatively, by chemiluminescence.