Whole-cell protein extracts were isolated using RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Thermo Scientific). Ten micrograms of total proteins were loaded and fractionated by SDS-PAGE, transferred to nitrocellulose membranes (Merck Millipore, Darmstadt, Germany), and probed with primary antibodies against pSTAT3, total STAT3 (rabbit, 1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (rabbit, 1:1000; Cell Signaling Technology). Immunoreactivity was visualized using SuperSignal West Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The protein extracts from αTC1 cells treated with IL-6 were used as a positive control. The expression levels of pSTAT3 were normalized to those of total STAT3.
Supersignal west extended duration substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperSignal West Extended Duration Substrate is a chemiluminescent substrate used for the detection of proteins in Western blotting applications. It provides a longer signal duration compared to standard chemiluminescent substrates, allowing for extended exposure times and increased sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Total stat3, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Nupage 10 bis tris, by Thermo Fisher Scientific (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Gel doc apparatus, by Bio-Rad (1 mentions)
4 protocols using supersignal west extended duration substrate
1
Quantification of STAT3 Phosphorylation
2
Immunoblotting of pSTAT3 and STAT3
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Whole-cell protein extracts were isolated using RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA) containing protease inhibitor cocktail (Thermo Scientific). Ten micrograms of total proteins was loaded and fractionated by SDS-PAGE, transferred to nitrocellulose membranes (Merck Millipore, Darmstadt, Germany), and probed with primary antibodies against pSTAT3, total STAT3 (rabbit, 1:1000; Cell Signaling Technology), and GAPDH (rabbit, 1:1000; Cell Signaling Technology). Immunoreactivity was visualized using SuperSignal West Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.
3
Western Blot Analysis of CAP1 in Breast Cancer
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The breast cancer cell lysates were prepared using RIPA buffer with protease and phosphatase inhibitors and protein concentration determined, as described previously (19 (link)). Equal amounts of protein samples (20 μg) were separated by pre-cast SDS-PAGE (NuPAGE 10% Bis-Tris, Invitrogen) and transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) milk in Tris-buffered saline Tween-20 (TBST) and probed with antibodies to CAP1 (1:750) or GAPDH (1:1000) as a loading control. Protein abundance was detected with horseradish peroxidase specific secondary antibodies and visualized by SuperSignal West Extended Duration Substrate (ThermoFisherScientific) using the Alpha Innotech FluorChem® FC2 imaging system and AlphaView version 3.0.3.0 software (ProteinSimple) or ImageJ software.
4
Western Blot Analysis of FTO Protein in Testis Cells
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Human testis biopsy whole cell extracts were mixed with 10% 1,4-dithiothreitol (Cleland's reagent) (DTT) (1 M), 1 Â lithium dodecyl sulfate, and distilled H 2 O, heat-denatured at 90 C for 10 minutes and then cooled on ice. The mixtures containing 10 and 20 mg of protein of whole cell extract were loaded on a 12% NuPAGE Gel (Life Technologies), electrophorized at 200 V for approximately 45 minutes, and transferred to a nitrocellulose membrane using a Transblot-Turbo blotter (BioRad) for 15 minutes. The membrane was blocked at room temperature for approximately 1 hour in PBST-M (5% milk in phosphate-buffered saline with 0.05% Tween 20) on a shaker. Primary anti-FTO antibody (1:3,000 in PBST-M), as previously reported ( 14) was added and incubated for 1 hour at room temperature on a shaker. Three times of 10-minute PBST washes followed. Secondary horseradish peroxidaseconjugated goat anti-rabbit antibody (catalogue no. Ab6721, AbCam) was diluted 1:40,000 in PBST-M and incubated at room temperature for 1 hour on a shaker. Three times of 10-minute PBST washes followed. The membrane was washed for 5 minutes in PBS. SuperSignal West Extended Duration Substrate (ThermoScientific) was added to the membrane. The bands were visualized with Gel Doc apparatus (Bio-Rad).
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