Goat anti rabbit antibody

Clarified cell lysate was diluted in denaturing SDS gel loading buffer, and boiled for 15 min. After denaturing, the samples were loaded onto a 4% to 12% gel for SDS-PAGE and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween 20, and then incubated with the following primary antibodies: anti-Flag antibody (Genscript, A00187), anti-Myc antibody (Genscript, A00172), anti-GST antibody (Genscript, A00097), or anti-TfR1 antibody (Santa Cruz Biotechnology, sc-32272). Then, the PVDF membrane was washed three times with PBS and incubated with HRP-conjugated Goat anti-Mouse antibody (Genscript, A00160) and Goat anti-Rabbit antibody (Genscript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).

The supernatant of cell lysates was diluted in denaturing buffer and boiled for 15 min. After denaturing, the samples were loaded onto a 4%–12% SDS-PAGE gel (Genscript) and separated by electrophoresis. Proteins were transferred to a PVDF membrane (Merck-Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween-20, and then incubated with the primary antibodies: anti-Flag antibody (1:1000, Genscript, A00187), anti-Myc antibody (1:1000, Genscript, A00172), or anti-GST antibody (1:1000, Genscript, A00097). Then, the membrane was washed three times with PBS and incubated with HRP-conjugated goat anti-mouse antibody (1:10,000, Genscript, A00160) and goat anti-rabbit antibody (1:10,000, Genscript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).

Clarified cell lysate was diluted in denaturing SDS gel loading buffer, and boiled for 15 min. After denaturing, the samples were loaded onto a 4 to 12% gel (GenScript, M41210C) for SDS-PAGE and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween 20, and then incubated with the following primary antibodies: anti-Flag antibody (GenScript, A00187), anti-Myc antibody (GenScript, A00172), anti-GST antibody (GenScript, A00097), or anti-His antibody (GenScript, A00174). The PVDF membrane was then washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (GenScript, A00160) and goat anti-rabbit antibody (GenScript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).