Western blot analyses were performed as previously described [28] (link). Cells were treated with NTHi for 30 min. Whole-cell lysate was separated in a 10% SDS-PAGE gel, transferred to polyvinylidene fluoride membrane, then incubated with antibodies against phospho-ERK1/2, total ERK1/2, phospho-MEK1/2, total MEK1/2, or β-actin. Respective proteins were visualized by using secondary HRP-conjugated rabbit or mouse IgG antibody (Santa Cruz Biotechnology) and the ECL detection system (Amersham ECL Prime Western Blotting Detection Regent, GE Healthcare).
Amersham ecl prime western blotting detection regent
Manufactured by GE Healthcare
Sourced in United States
Amersham ECL Prime Western Blotting Detection Reagent is a chemiluminescent detection reagent designed for use in Western blotting analysis. It is used to detect and visualize target proteins on a membrane after separation by gel electrophoresis and transfer.
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2 protocols using amersham ecl prime western blotting detection regent
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Western Blot Analysis of Cellular Signaling
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Western Blot Protein Detection
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Whole cell lysates were separated on 10% SDS–PAGE gels and transferred to polyvinylidene difluoride membranes (PVDF). The membrane was blocked with 5% skim milk (Neogen Co., Lansing, MI, USA) and then incubated with a 1:1000 diluent of a primary antibody in TBS with 5% BSA. The membrane was washed 5 times in TBS-T and then incubated with 1:5000 diluent of the corresponding secondary antibodies HRP-conjugated IgG antibody (Cell Signaling Technology, Danvers, MA, USA) in TBS with 5% skim milk. Target proteins were visualized by using the ECL detection system (Amersham ECL Prime Western blotting Detection Regent, GE Healthcare, Chicago, IL, USA).
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