Cells were lysed in RIPA buffer and 20 μg of protein lysate were separated on NuPAGE 4–12% Bis-Tris Midi Gels (Life Technologies, Cat. # WG1402BOX). Proteins were transferred onto nitrocellulose membrane and stained with Ponceau Red to confirm complete transfer. Membranes were blocked in 5% non-fat dry milk in TBS-T and incubated over night at 4°C with anti-GFP (1:1,000; Cell Signalling Technologies, Cat. # 2956) or anti-Actin (1:4,000; Invitrogen, Cat. # AM4302) antibodies diluted in 5% non-fat dry milk in TBS-T. The membranes were washed with TBS-T and incubated with HRP-conjugated secondary anti-rabbit antibody (1:20,000; Jackson ImmunoResearch, Cat. # 115-035-003) for 1 hour. The signal was detected with SuperSignal West Pico PLUS Chemoluminescent Substrate (Thermo Scientific, Cat. #34577) following the manufacturer’s instructions.
Supersignal west pico plus chemoluminescent substrate
Manufactured by Thermo Fisher Scientific
SuperSignal West Pico PLUS Chemoluminescent Substrate is a laboratory reagent used for the detection of proteins in Western blot analysis. It generates a chemiluminescent signal that can be detected by imaging equipment, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
2 protocols using supersignal west pico plus chemoluminescent substrate
1
Western Blot Analysis of GFP and Actin
2
Western Blot Protein Analysis
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Cells were lysed in RIPA buffer and 20 μg of protein lysate were separated on NuPAGE 4-12% Bis-Tris Midi Gels (Invitrogen). Proteins were transferred onto nitrocellulose membrane and stained with Ponceau Red to confirm complete transfer. Membranes were blocked in 5% non-fat dry milk in TBS-T and incubated over night at 4°C with anti-GFP (1:1,000; Cell Signaling Technologies) or anti-Actin (1:4,000; Invitrogen) antibodies diluted in 5% non-fat dry milk in TBS-T. The membranes were washed with TBS-T and incubated with HRP-conjugated secondary anti-rabbit antibody (1:20,000; Jackson ImmunoResearch) for 1 hour. The signal was detected with SuperSignal West Pico PLUS Chemoluminescent Substrate (Thermo Scientific) following the manufacturer's instructions.
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