On the day of the analysis, cell lysates were thawed on ice and then combined with 2x Laemmli loading Buffer (BioRad) with 0.05% BME. The samples were heat denatured for 10 min at 95 °C and loaded into a Bolt™ 4 to 12% (vol/vol), Bis-Tris precast polyacrylamide gels (Invitrogen by Thermo Fisher). Gels were run at 120V in 1x MOPS Buffer and were then transferred to a 0.2 μm PVDF nitrocellulose membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) using the Trans-Blot Turbo Transfer System (13A, 25V, 7 min) (BioRad). The membrane was blocked in 5% BSA for 1 h followed by overnight incubation at 4 °C with primary antibodies (1:1000 anti-mCherry [Cell Signaling Technology] or 1:1000 anti-DYKDDDDK [Cell Signaling Technology]). Membranes were washed with TBST (3x, 5 min) and incubated with secondary anti-rabbit HRP antibody for 1 h at room temperature followed by a subsequent washing with TBST (3x, 5 min). The membrane was developed with ECL western blotting detection reagent kit (Thermo Scientific) and imaged on ChemiDoc XRS+ with Image Lab Software (Bio-Rad).
Bis tris precast polyacrylamide gels
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
Bis-Tris precast polyacrylamide gels are laboratory equipment used for electrophoresis to separate and analyze proteins. They are pre-cast, ready-to-use gels that utilize a Bis-Tris buffering system for protein separation.
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Lab products found in correlation
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Laemmli loading buffer, by Bio-Rad (1 mentions)
Anti dykddddk, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection reagent kit, by Thermo Fisher Scientific (1 mentions)
Anti mcherry, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Anti irf9, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti irf2, by Abcam (1 mentions)
Anti yy1, by Cell Signaling Technology (1 mentions)
2 protocols using bis tris precast polyacrylamide gels
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis by Western Blot
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Protein extracts were prepared using RIPA buffer (Sigma: 150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mM Tris, pH8.0) completed with Protease Inhibitor (PI) Cocktail tablet (Sigma). Protein concentration was determined with the Pierce BCA protein assay kit (Thermo Scientific). Protein extracts (20-40 µg) were fractionated by SDS polyacrylamide gel electrophoresis on 4–12% Bis–Tris precast polyacrylamide gels (Invitrogen, Paisley, UK) and transferred to a nitrocellulose membrane (Invitrogen). Bands were visualized by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK). Protein quantification was performed using ImageJ and statistical significance was assessed using GraphPad Prism 8. Anti-IRF2 (Abcam, cat#ab124744, 1:500 dilution), anti-IRF9 (Cell Signaling, cat#76684, 1:500 dilution), anti-YY1 (Cell Signaling, cat#2185, 1:500 dilution), anti-SNAI2 (Abcam, cat#ab27568, 1:200 dilution), and anti-p16 (CDKN2A, Abcam, cat#ab54210, 1:1000 dilution) antibodies were used.
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