To evaluate the expression of target proteins of transfected cells, we collected the cells for lysis in RIPA buffer (Beyotime, Shanghai, China) on ice. Protein samples of the same amount were loaded in 10% SDS-PAGE for electrophoresis separation and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Massachusetts, USA). While blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated at 4 °C overnight with primary antibodies including FGFR1 (diluted 1:1000, CST, #9740), E-cadherin (diluted 1:1000, CST, #3195), N-cadherin (diluted 1:1000, CST, #13,116), Vimentin (diluted 1:1000, CST, #5741), ID4 (diluted 1:1000, Abcam, ab220166), SMAD6 (diluted 1:1000, ab273106), SMAD7 (diluted 1:1000, ab216428) and GAPDH (diluted 1:1000, Beyotime, AG019). When washed with Tris-buffered saline containing 0.05% Tween-20 (TBST buffer) for three times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000), the protein bands were visualized by chemiluminescence reagents (P10100, NcmECL Ultra).
Ab220166
Manufactured by Abcam
Sourced in United Kingdom
Ab220166 is a polyclonal antibody produced in rabbit that targets the human Tenascin-C protein. This antibody can be used in various immunoassay applications to detect and quantify Tenascin-C expression.
Automatically generated - may contain errors
2 protocols using ab220166
1
Western Blot Analysis of Transfected Cells
2
Western Blot Protein Analysis Protocol
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Briefly, after cell lysis with RIPA + a protease inhibitor, centrifugation of the
lysate was performed at 4°C for 10 min at 12 000 r/min. The obtained protein
samples were quantitated with a BCA kit (Thermo Fisher Scientific, MA, USA),
boiled at 95°C, and sequentially separated by SDS-PAGE, which were loaded to
nitrocellulose membranes. Then, membranes were silked in 5% skimmed milk.
Membranes were reacted with primary antibodies (ID4 [ab220166, 1:1000, Abcam,
Cambridge, UK] and GAPDH [ab181602, 1:10 000, Abcam, Cambridge, UK]) overnight.
Later, they were cultured with horseradish peroxidase (HRP)-conjugated secondary
antibody goat anti-rabbit IgG (ab6721, 1:3000, Abcam, Cambridge, UK) for
120 min. At last, an enhanced chemiluminescence detection Kit (Solarbio,
Beijing, China) was used for protein band visualization.
lysate was performed at 4°C for 10 min at 12 000 r/min. The obtained protein
samples were quantitated with a BCA kit (Thermo Fisher Scientific, MA, USA),
boiled at 95°C, and sequentially separated by SDS-PAGE, which were loaded to
nitrocellulose membranes. Then, membranes were silked in 5% skimmed milk.
Membranes were reacted with primary antibodies (ID4 [ab220166, 1:1000, Abcam,
Cambridge, UK] and GAPDH [ab181602, 1:10 000, Abcam, Cambridge, UK]) overnight.
Later, they were cultured with horseradish peroxidase (HRP)-conjugated secondary
antibody goat anti-rabbit IgG (ab6721, 1:3000, Abcam, Cambridge, UK) for
120 min. At last, an enhanced chemiluminescence detection Kit (Solarbio,
Beijing, China) was used for protein band visualization.
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