Western blot ecl plus kit

Protein samples were diluted in SDS-PAGE sample buffer including β-mercaptoethanol. The samples were boiled for 5 min and subjected to SDS-PAGE in a 10 % (w/v) gel. After electrophoresis, the gel was either stained with Coomassie brilliant blue or the proteins were transferred from the gel onto a Hybond-P membrane (GE Healthcare, USA) by electroblotting. To prevent nonspecific binding of antibodies with the membrane, it was treated with 5 % (w/v) solution of dry milk in TBS-T (150 mM NaCI, 20 mM Tris, 0.1 % Tween 20, pH 8.0) buffer. The membrane was probed with mouse monoclonal antibodies specific for the M2e peptide and then incubated with rabbit anti-mouse antibodies conjugated with peroxidase. Specific protein complexes were detected using a Western Blot ECL Plus kit (GE Healthcare, USA). The intensity of the bands in stained gels and blots was determined using Nonlinear. Dynamics. TotalLab. TL120.v2009-NULL software.
Western blot analysis of purified Flg-4M under non-reducing conditions was performed as above but that β-mercaptoethanol was not included in the sample buffer and the sample was not boiled before loading onto a gel.

The leaf tissue in the agroinfiltrated area was homogenized with a mortar and pestle and mixed with an equal volume of 2× SDS-PAGE loading buffer (20% glycerol, 5% SDS, 62.5 mM Tris-HCI pH 6.8, 0.5% bromphenol blue, 5% β-mercaptoethanol). The purified proteins were diluted two-fold in the same buffer. The samples were boiled for 10 min and subjected to SDS-PAGE in a 10% (w/v) gel. After electrophoresis, the gel was either stained with Coomassie brilliant blue or the proteins were transferred from the gel onto a Hybond-P membrane (GE Healthcare, Chicago, IL, USA) by electroblotting. To prevent the nonspecific binding of antibodies with the membrane, it was treated with a 5% (w/v) solution of dry milk in TBS-T (150 mM NaCI, 20 mM Tris, 0.1 % Tween 20, pH 8.0) buffer. The membrane was probed with mouse monoclonal antibodies specific for the M2e peptide (Anti-Influenza A Virus M2 Protein antibody [14C2] (ab5416); Abcam, Cambridge, UK) and then incubated with Anti-Mouse IgG HRP Conjugate (W4021, Promega). Specific protein complexes were detected using a Western Blot ECL Plus kit (GE Healthcare).

Protein extracts isolated from plant tissues were mixed with SDS-PAGE sample buffer containing β-mercaptoethanol. The samples were boiled for 10 min and subjected to SDS-PAGE on 15% (w/v) gels. After electrophoresis, gels were either stained with Coomassie brilliant blue or the proteins were transferred from the gel onto a Hybond-P membrane (GE Healthcare, USA) by electroblotting. To prevent non-specific binding of antibodies with the membrane, it was treated with 5% (w/v) solution of dry milk in TBS-T (150 mM NaCI, 20mM Tris, 0.1% (v/v) Tween 20, pH 8.0) buffer. The membrane was probed with mouse monoclonal antibodies specific for the M2e peptide and then incubated with rabbit anti-mouse secondary antibodies conjugated with peroxidase. Specific immunoreactive proteins were detected using a Western Blot ECL Plus kit (GE Healthcare, USA). The intensity of the bands in stained gels and blots was determined using Nonlinear.Dynamics.TotalLab.TL120.v2009-NULL software.