Cell extracts were prepared with RIPA buffer (Cell Signaling Technology, 9806) as a protein extraction reagent plus a protease/phosphatase inhibitor cocktail (Cell Signaling Technology, 5872). The cell lysates were separated on a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gel and transferred with a Trans-Blot Turbo RTA Mini 0.2 μm Nitrocellulose Transfer Kit (Bio-Rad, 1704270). The membranes were blocked with blocking buffer (VWR international, AAJ60473AP) at room temperature for 2 hours, then incubated with primary antibody at 4°C overnight. The following primary antibodies were used: MT1-MMP (Abcam, ab51074, 1:5000), MRC2 (Abcam, ab71032, 1:1000), CD276 (Abcam, ab219648, 1:1000), Cofilin (Abcam, ab124979, 1:1000), and β-actin (Cell Signaling Technology, 4967, 1:1500). The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology Cat# 7074, RRID:AB_2099233, 1:1000) at room temperature for 1 hour. Western blotting was performed with Clarity Western ECL substrate (Bio-Rad, #170–5060). The ChemiDoc System (Bio-Rad) was used for images.
Trans blot turbo rta mini 0.2 μm nitrocellulose transfer kit
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot Turbo RTA Mini 0.2 μm Nitrocellulose Transfer Kit is a laboratory equipment designed for the rapid and efficient transfer of proteins from polyacrylamide gels to nitrocellulose membranes. It utilizes a rapid transfer assembly (RTA) system to facilitate the transfer process.
Automatically generated - may contain errors
Lab products found in correlation
Ab51074, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Wedgewell gel, by Thermo Fisher Scientific (1 mentions)
Rabbit anti np polyclonal antibody, by GeneTex (1 mentions)
2 protocols using trans blot turbo rta mini 0.2 μm nitrocellulose transfer kit
1
Western Blot Analysis of Cell Signaling Proteins
2
Profiling Baculovirus-Infected Insect Cell Proteins
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Culture media from NoV-M2/NP baculovirus-infected Sf9 insect cells was harvested 96 h post-infection. Culture media from cells infected with an empty NoV platform was used as a control. Samples were loaded on 10%–20% precast WedgeWell Gel (Thermo Fisher Scientific, Waltham, USA) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto nitrocellulose membranes was performed using Trans-Blot Turbo RTA Mini 0.2 μm Nitrocellulose Transfer Kit (Bio-Rad, Hercules, USA). Membranes were then blocked for 1 h in a 5% semi-skimmed milk solution (5% milk/TBS/0.01% Tween 20) and incubated overnight with primary antibodies solution: rabbit polyclonal anti-NoV serum (obtained by vaccination of rabbit with insect cell-derived NoV VP1), mouse monoclonal anti-M2 (Santa Cruz Biotechnologies, Dallas, USA) rabbit anti-NP polyclonal antibody (Genetex, Irvine, USA), or anti-β-actin antibodies (Abcam, Waltham, USA) in 5% milk/TBS/0.01% Tween 20. Then, the membranes were washed (TBS/0.01% Tween 20) and incubated with a solution of AffiniPure goat anti-rabbit or anti-mouse antibodies (Jackson Immuno Research, Ely, United Kingdom) in 5% milk/TBS/0.01% Tween 20. The reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, USA).
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