Protein expression was analyzed from total cell lysate. 1.2 × 106 cells were lysed in SDS/PAGE sample buffer (200 µl 2× sample buffer, 245 µl H2O, 50 µl SDS [20% wt/vol], and 5 µl DTT), boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, and then incubated with appropriate primary antibodies. All primary antibodies were used at 1:1,000 dilution unless otherwise noted. Rabbit anti-BAF (ab129184; Abcam), rabbit anti–lamin A/C (2032S; Cell Signaling Technology), rabbit anti–LEMD2 (HPA017340; Atlas Antibodies), rabbit anti-Emerin (2659S; Cell Signaling Technology), rabbit anti-Ankle2 (ab225905; Cell Signaling Technology), and rabbit anti-Chmp7 (16424-1-AP; Proteintech) were used to verify siRNA knockdown efficiency. Mouse monoclonal anti-tubulin was used as a loading control (1:2,000; sc-32293; Santa Cruz Biotechnology). Primary antibodies were detected using HRP-conjugated anti-rabbit (1:5,000; G21234; Thermo Fisher Scientific) or anti-mouse (1:5,000; F21453; Thermo Fisher Scientific) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via the Bio-Rad ChemiDoc MP Imaging System.
F21453
Manufactured by Thermo Fisher Scientific
Sourced in United States
The F21453 is a centrifuge designed for general laboratory use. It has a maximum speed of 6,000 RPM and a maximum capacity of 4 x 100 mL. The centrifuge is constructed with a sturdy metal housing and features a brushless motor for reliable operation.
Automatically generated - may contain errors
Lab products found in correlation
Sc 32293, by Santa Cruz Biotechnology (5 mentions)
G 21234, by Thermo Fisher Scientific (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Mini protean tgx, by Bio-Rad (5 mentions)
Ab129184, by Abcam (4 mentions)
Ab225905, by Cell Signaling Technology (3 mentions)
Hpa017340, by Atlas Antibodies (3 mentions)
Ab290, by Abcam (3 mentions)
Odyssey fc imaging system, by LI COR (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ab7403, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
5 protocols using f21453
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Nuclear Proteins
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Total cell lysates were used to analyze protein expression. 1.2 × 106 cells were lysed using SDS-PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient (Mini-PROTEAN TGX; Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, and then incubated with appropriate primary antibodies: rabbit anti-LaA/C (1:1000; 2032S; Cell Signaling Technology), rabbit anti-GFP (1:1000; ab290; Abcam), rabbit anti–BAF (1:1000; ab129184; Abcam), rabbit anti–LEMD2 (1:1000; HPA017340; Atlas Antibodies, Bromma, Sweden), rabbit anti-Emerin (1:1000; 2659S; Cell Signaling Technology), and rabbit anti-Ankle2 (1:1000; ab225905; Cell Signaling Technology). Mouse monoclonal anti-tubulin (1:5000; sc-32293; Santa Cruz Biotechnology) was used as a loading control. The primary antibodies were detected using horseradish peroxidase (HRP)–conjugated anti-rabbit (1:5000; G21234; Thermo Fisher Scientific) or anti-mouse (1:5000; F21453; Thermo Fisher Scientific) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via a LI-COR ODYSSEY Fc Imaging system (LI-COR Biosciences, Lincoln, NE, USA).
3
Immunoblot Analysis of Cell Lysates
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To analyze total cell lysates by immunoblot, 1.2 × 106 cells were lysed in SDS–PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, US). After blocking with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, the membrane was incubated with appropriate primary antibodies: rabbit polyclonal anti-hemagglutinin (1:2000; Ab9110; Abcam, Cambridge, UK) and mouse monoclonal anti-tubulin as a loading control (1:10000; sc-32293; Santa Cruz Biotechnology, Dallas, TX, USA). The primary antibodies were detected using horseradish peroxidase (HRP)–conjugated anti-rabbit (1:40,000; G21234; Thermo Fisher Scientific, Waltham, MA, USA) or anti-mouse (1:40,000; F21453; Thermo Fisher Scientific, Waltham, MA, USA) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via a Bio-Rad ChemiDoc MP System (Bio-Rad, Hercules, CA, USA). Following detection of each antibody, the membrane was quenched with 30% H2O2 for 30 min. To detect biotinylated proteins, the membrane was incubated with HRP-conjugated streptavidin (1:40,000; ab7403; Abcam, Cambridge, UK) in 0.2% Triton X-100 in PBS for 45 min.
4
Protein Expression Analysis by Western Blot
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Protein expression was analyzed from total cell lysate. 1.2 × 106 cells were lysed in SDS/PAGE sample buffer (200 μL 2x sample buffer, 245 μL H2O, 50 μL SDS (20% w/v), and 5 μL DTT), boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4–20% gradient gels (Mini-PROTEAN TGX; Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, then incubated with appropriate primary antibodies. Primary rabbit anti-BAF (ab129184; Abcam) was used at a dilution of 1:1000. Rabbit anti-GFP (ab290; Abcam) was used at a dilution of 1:10000. Mouse monoclonal anti-tubulin was used as a loading control (sc-32293; Santa Cruz Biotechnology) at 1:2000 dilution. Primary antibodies were detected using HRP-conjugated anti-rabbit (1:5000; G21234; ThermoFisher), or anti-mouse (1:5000; F21453; ThermoFisher) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via BioRad ChemiDoc™ MP Imaging System (BioRad, Hercules, CA).
5
Western Blot Analysis of Nuclear Proteins
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+ Expand
Total cell lysates were used to analyze protein expression. 1.2 x 106 cells were lysed using SDS-PAGE sample buffer, boiled for 5 min, and sonicated to shear DNA. Proteins were separated on 4-20% gradient (Mini-PROTEAN TGX; Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). Membranes were blocked with 10% (vol/vol) adult bovine serum and 0.2% Triton X-100 in PBS for 30 min, and then incubated with appropriate primary antibodies: rabbit anti-LaA/C (1:1000; 2032S; Cell Signaling Technology), rabbit anti-GFP (1:1000; ab290; Abcam), rabbit anti-BAF (1:1000; ab129184; Abcam), rabbit anti-LEMD2 (1:1000; HPA017340; Atlas Antibodies), rabbit anti-Emerin (1:1000; 2659S; Cell Signaling Technology), and rabbit anti-Ankle2 (1:1000; ab225905; Cell Signaling Technology). Mouse monoclonal anti-tubulin (1:5000; sc-32293; Santa Cruz Biotechnology) was used as a loading control. The primary antibodies were detected using horseradish peroxidase (HRP)-conjugated anti-rabbit (1:5,000; G21234; Thermo Fisher Scientific) or anti-mouse (1:5,000; F21453; Thermo Fisher Scientific) antibodies. The signals from antibodies were detected using enhanced chemiluminescence via a LI-COR ODYSSEY Fc Imaging system (LI-COR Biosciences).
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