The cells lysates were mixed with Laemmli buffer containing 2-mercaptoethanol. Then, the mixtures were boiled at 95°C for 5 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (400 mA, 1 h). After membranes were incubated overnight with primary antibodies, they were washed two times for 10 min with 0.2% tris-buffered saline Tween. Next, membranes had been incubated with secondary antibodies for 1 h. Before 5-min incubation with chemiluminescent HRP substrate (Millipore Corporation, Billerica, U.S.A.), membranes were washed three times for 10 min with 0.2% tris-buffered saline Tween. Finally, bands were visualized by Chemidoc XRS System (Bio-Rad, Hercules, CA). The following antibodies were used: rabbit anti-AR (Abcam) and mouse anti-Actin (Servicebio).
Mouse anti actin
Manufactured by Wuhan Servicebio Technology
Sourced in China
The Mouse anti-Actin is a monoclonal antibody that specifically binds to the actin protein, a highly conserved cytoskeletal protein found in all eukaryotic cells. This antibody is commonly used in various research applications as a loading control or reference for Western blotting and other immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescent hrp substrate, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Wuhan Servicebio Technology (1 mentions)
Ecl plus, by Solarbio (1 mentions)
Mouse anti acox1 sc 517306, by Santa Cruz Biotechnology (1 mentions)
Alphaview 3, by Bio-Techne (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti sox9, by Abcam (1 mentions)
4 protocols using mouse anti actin
1
Western Blot Analysis of Androgen Receptor
2
Adipocyte Protein Extraction and Western Blot
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The total protein of adipocyte samples was extracted by using RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (Servicebio, Wuhan, China) (100:1). Proteins in cell lysates were separated by 10% SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (ISEQ00010, Millipore, Boston, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h and then incubated with mouse anti-ACAA1 (sc-514051, Santa Cruz, Dallas, TX, USA), and mouse anti-ACOX1 (sc-517306, Santa Cruz, Dallas, TX, USA) antibodies at 4 °C overnight, followed by incubation with a secondary antibody conjugated with horse radish peroxidase (HRP) (GB23303, Servicebio) for 1 h at room temperature. Mouse anti-actin (Servicebio,) was used as the internal control. Signals were enhanced by ECL Plus (Solarbio, Beijing, China), and images were captured and analyzed by Photoshop CS6 and AlphaView 3.0 (Alpha Innotech, San Jose, CA, USA).
3
Protein Expression Analysis via Simple Western Blotting
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Protein content analysis was performed using Simple Western™ Automated Western Blot Systems (Protein Simple, San Jose, CA, USA) using a 12–230 kDa separation module (SM-W004) and anti-rabbit detection module (DM-001) according to the manufacturer’s instructions (Mishra et al., 2017 (link); Castle et al., 2019 (link)). Briefly, CCs were lysed with lysis buffer, and the protein concentration was determined using the BCA protein assay (Thermo Scientific, Waltham, MA, USA). Lysate was loaded into designated wells in a 25-well plate followed by electrophoresis. The primary antibodies used were mouse anti-actin (Servicebio, Wuhan, China), 1:3000; rabbit anti-PHGDH (Servicebio, Wuhan, China), 1:750; rabbit anti-PSAT1 (Abcam, Cambridge, MA, USA), 1:1000; rabbit anti-PSPH (ABclonal, Wuhan, China), 1:750; and rabbit anti-SHMT2 (Servicebio, Wuhan, China), 1:750. The relative expression of the protein was corrected according to that of actin as the internal reference. The digital image was analysed using Compass software (ProteinSimple, San Jose, CA, USA), and the quantified data of the detected protein were reported as molecular weight and signal/peak intensity.
4
Western Blot Analysis of SOX9 Protein
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The cells lysates mixed with Laemmli buffer containing 2-mercaptoethanol were boiled for 5 min at 99℃. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 200 V, 45 min) and then transferred onto a nitrocellulose membrane (400 mA, 1 h). Membranes were incubated overnight with primary antibodies and washed 3 times for 10 min with 0.2% tris-buffered saline Tween. Then, membranes had been incubated with secondary antibodies for 1 h and washed 3 times for 10 min with 0.2% tris-buffered saline Tween. Next, 5-min incubation between chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA) and membranes were conducted and bands were visualized by Chemidoc XRS System (Bio-Rad, Hercules, CA). The following antibodies were used: rabbit anti-SOX9 (Abcam) and mouse anti-Actin (Servicebio).
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