30 μg proteins lysed from the transfected cells were subjected to SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (Roche). The anti-RFP antibody (Abcam, Cambridge, UK) was used, and immunoblotting with anti-β-tubulin antibody (Proteintech, Chicago, IL, USA) was conducted as internal control. The signals were measured using an Enhanced Chemiluminescence (ECL) western blot detection kit (Sangon).
Anti β tubulin antibody
Manufactured by Proteintech
Sourced in United States
The Anti-β-tubulin antibody is a primary antibody that specifically recognizes the β-tubulin protein, a key component of the cytoskeletal microtubules in eukaryotic cells. This antibody can be used to detect and visualize the distribution of β-tubulin in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Anti rfp antibody, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Roche (1 mentions)
Ecl western blot detection kit, by Sangon (1 mentions)
Anti p21 antibody, by Proteintech (1 mentions)
Ecl western blotting detection reagent, by Thermo Fisher Scientific (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Proteintech (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoctm touch imaging system, by Bio-Rad (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Second antibody, by Proteintech (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Beyoecl plus reagent, by Beyotime (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Anti p p70s6k, by Cell Signaling Technology (1 mentions)
Anti p eif2a, by Cell Signaling Technology (1 mentions)
6 protocols using anti β tubulin antibody
1
Quantitative Western Blot Analysis
2
CDK1 Protein Expression Analysis
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We seeded 1 × 105 cells in 6-well plates. Cells were then collected at 48 h post-transfection and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech, Beijing, China). Twenty micrograms of proteins were electrophoresed on a SDS polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Roche). Anti-CDK1 polyclonal antibodies (GeneTex, San Antonio, TX, USA) were used, and immunoblotting with anti-β-tubulin antibody (Proteintech, Rosemount, IL, USA) was conducted to ensure equal protein loading. Signals were then measured by an enhanced chemiluminescence (ECL) western blot detection kit (Sangon Biotech).
3
Western Blot Analysis of p21 and β-Tubulin
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Western blot analysis was carried out as described by us previously31 (link)33 (link). The antibodies used in this study include an anti-p21 antibody (1:500 dilution, Proteintech, Wuhan, China) and an anti-β-tubulin antibody (1:2000 dilution, Proteintech, Wuhan, China).
4
Western Blot Assay Antibody Protocol
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Following antibodies were used in for Western blot assays in this paper: anti-Flag antibody (Sigma-Aldrich, F1804, 1:1000 used), anti-β-tubulin antibody (ProteinTech, 66240-1-Ig, 1:2000 used), anti-Lamin B1 (ProteinTech, 66095-1-Ig, 1:1000 used), anti-GAPDH (60004-1-Ig, 1:5000 used), HRP-conjugated anti-mouse secondary antibody (Cell Signaling Technology, 7076, 1:3000 used) and HRP-conjugated anti-rabbit secondary antibody (ProteinTech, SA00001-2, 1:3000 used). The cell pellets after transfection were lysed in ice-cold lysis buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 400 mM NaCl, 10 mM KCl, 1% Nonidet P-40 and 20% Glycerol) containing protease inhibitor cocktail and PMSF. After centrifugation at 12,000 × g for 15 min, the supernatant separated by 10% SDS-PAGE, transferred to PVDF membranes (Thermo Scientific). After an 1 h incubation with blocking buffer (5% BSA), the membrane was incubated with corresponding antibodies overnight at 4 °C. The membrane was washed and incubated with HRP-conjugated secondary antibodies. The membranes were washed again, developed with ECL Western Blotting Detection Reagents (Thermo Scientific) and visualised with ChemiDocTM Touch Imaging System (Bio-Rad).
5
Western Blot Protein Detection Protocol
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Whole‐cell lysates were extracted using RIPA lysis buffer (Beyotime) on ice for 30 min. Then the supernatants were collected by centrifugation (1400 g for 15 min at 4°C). Sample protein concentrations were quantified by protein BCA Assay (Thermo Scientific). The protein samples were electrophoresed in 12% SDS‐PAGE after boiling for 10 min, and then transferred to a PVDF membrane. The membrane was blocked in 5% bovine serum albumin (BSA) at room temperature for 2 h, and then incubated overnight with primary anti‐PLEK2 antibody (1:1000, Abclonal) and anti‐β‐tubulin antibody (1:1000, Proteintech) at 4°C. After 3 washes in TBST (10 min each), the membrane was incubated with the second antibody (1:5000, Proteintech) at room temperature for 2 h, followed by 3 washes in TBST. The protein bands were visualized using BeyoECL Plus reagent (Beyotime).
6
Protein Expression Analysis in Bovine Muscle
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Approximately 50 mg of frozen BF muscle was taken and homogenized in 200 μL of ice-cold RIPA lysis buffer (Solarbio, Beijing, China) containing 1 mmol/L PMSF, 5 mmol/L NaF and 1 mmol/L Na3VO4. The homogenates were shaken on ice for 30 min and then centrifuged (12,000 × g at 4 °C for 15 min) to obtain the supernatant. Ten microliters of supernatant was taken to determine protein concentration using a BCA Protein Assay kit (Tiangen, Beijing, China). The remaining supernatant was diluted to the proper protein concentration with RIPA buffer and then mixed with 5 × loading buffer (CWBIO, Beijing, China) and finally boiled at 100 °C for 10 min to obtain the samples.
The protein expression was measured by Western blot analysis as described previously (Guo et al., 2018 (link)). Beta-tubulin was chosen as the reference protein, and the anti-β-tubulin antibody (1:2,000; catalog number 10094-1-AP) was purchased from Proteintech Group (Rosemont, IL 60018, USA). The antibodies including anti-AMPKα, anti-p-AMPKα, anti-p70S6K, anti-p-p70S6K, anti-eIF2α and anti-p-eIF2a (1:100; catalog numbers 2532, 2531, 9202, 9205, 9722, and 9721, respectively) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies used were goat anti-rabbit or anti-mouse HRP-conjugated (1:4,000; Bioworld Technology).
The protein expression was measured by Western blot analysis as described previously (Guo et al., 2018 (link)). Beta-tubulin was chosen as the reference protein, and the anti-β-tubulin antibody (1:2,000; catalog number 10094-1-AP) was purchased from Proteintech Group (Rosemont, IL 60018, USA). The antibodies including anti-AMPKα, anti-p-AMPKα, anti-p70S6K, anti-p-p70S6K, anti-eIF2α and anti-p-eIF2a (1:100; catalog numbers 2532, 2531, 9202, 9205, 9722, and 9721, respectively) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies used were goat anti-rabbit or anti-mouse HRP-conjugated (1:4,000; Bioworld Technology).
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