Cellular protein was lysed by radio immune precipitation assay (RIPA) buffer (Beyotime, China) with phenylmethane sulfonyl fluoride (PMSF) protease inhibitor (Beyotime, China) and the homogenate was centrifuged at 12,000 × g for 5 min at 4 ◦C. The supernatant was collected and the protein concentration was determined immediately using a BCA protein quantification kit (Beyotime, China). The proteins were separated in 10% SDS-PAGE and transferred onto PVDF membrane, and then probed with antibodies following standard procedures. The antibodies and their dilutions utilized for western blots were as follow: anti-GHR (bs-0654R; Bioss, China; 1:500), anti-PGC1α (bs-1832R; Bioss, China; 1:500), anti-NRF1 (12,482–1-AP; Proteintech, USA; 1:500), anti-TOMM20 (AF1717; Beyotime, China; 1:500), anti-JAK2 (bs-0908R; Bioss, China; 1:1000), anti-p-JAK2 (bsm-52171R; Bioss, China; 1:1000), anti-AKT1 (bs-0115 M; Bioss, China; 1:500), anti-p-AKT1 (66,444–1-Ig; Proteintech, China; 1:500), anti-CREB1 (bs-0035R; Bioss, China; 1:500), anti-p-CREB1 (bs-0036R; Bioss, China; 1:500), anti-β-actin (bs-0061R; Bioss, China; 1:1000), goat anti-rabbit IgG-HRP (bs-0295G; Bioss, China; 1:5000), goat anti-mouse IgG-HRP (bs-0296G; Bioss, China; 1:5000).
Anti β actin bs 0061r
Manufactured by Bioss Antibodies
Sourced in China
Anti-β-actin (bs-0061R) is a primary antibody product from Bioss Antibodies. It is designed to detect and bind to the β-actin protein, which is a widely expressed cytoskeletal protein in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti p akt1, by Proteintech (1 mentions)
Bs 0296g, by Bioss Antibodies (1 mentions)
Protease inhibitor pmsf, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Goat anti mouse igg hrp, by Bioss Antibodies (1 mentions)
Anti pgc1α bs 1832r, by Bioss Antibodies (1 mentions)
Sa00004 10, by Proteintech (1 mentions)
Bca kit, by Beyotime (1 mentions)
Image lab software, by Shanghai Tanon Science & Technology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Wuhan Servicebio Technology (1 mentions)
Lysis buffer, by Wuhan Servicebio Technology (1 mentions)
Bs 0295g, by Bioss Antibodies (1 mentions)
Lysozyme from chicken egg white, by Merck Group (1 mentions)
Anti phospho eif2α 3398, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Beyotime (1 mentions)
5 protocols using anti β actin bs 0061r
1
Western Blot Analysis of Cellular Signaling
2
Exosomal Protein Analysis by Western Blot
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Protein from EC9706-Exos was extracted and measured with BCA kit (Beyotime
Biotechnology). Protein was transferred to PVDF membrane after 10% SDS-PAGE gel
electrophoresis. After enclosing with 5% skimmed milk, the antibodies including
anti-CD63 (bs-1523R, 1: 2, 000, Bioss), anti-TSG101 (bs-1365R, 1: 2, 000,
Bioss), and anti-β-actin (bs-0061R, 1: 2, 000, Bioss) were incubated overnight.
Secondary resistance (1: 4,000, SA00004-10, ProteinTech) was subsequently
incubated at 37 °C. Finally, ECL was utilized to detect protein blots, whereas
ImageJ software (NIH, version 4.3) was adopted for quantification (Table 2 ).
Biotechnology). Protein was transferred to PVDF membrane after 10% SDS-PAGE gel
electrophoresis. After enclosing with 5% skimmed milk, the antibodies including
anti-CD63 (bs-1523R, 1: 2, 000, Bioss), anti-TSG101 (bs-1365R, 1: 2, 000,
Bioss), and anti-β-actin (bs-0061R, 1: 2, 000, Bioss) were incubated overnight.
Secondary resistance (1: 4,000, SA00004-10, ProteinTech) was subsequently
incubated at 37 °C. Finally, ECL was utilized to detect protein blots, whereas
ImageJ software (NIH, version 4.3) was adopted for quantification (
3
In vivo Expression of FlaB Protein in Mouse Tumors
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To confirm the expression of FlaB protein in vivo, mouse tumor tissues were collected 24 h after l -arabinose injection, and total proteins were obtained from those tissues by adding ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer (Servicebio, Wuhan, China). Equivalent amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated with anti-His6 antibody (9F2) (1:4,000, Fujifilm Wako Pure Chemicals Industries, VA, USA) at −4°C overnight. Anti-β-actin (bs-0061r, Bioss Antibodies, Beijing, China, 1:5,000) was used as a housekeeping marker. The bands on the membrane were developed with a Super ECL Plus Kit (Boster Biological Technology, Wuhan, China) and recorded via Image Lab software (4600SF, Tanon Science & Technology, Xinjiang, China).
4
Chaperone-Assisted Enzymatic Assays
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Alcohol dehydrogenase lyophilized powder sourced from baker’s yeast was obtained from SRL. CS and MDH sourced from the porcine heart (Sigma), and lysozyme from chicken egg white (Sigma) were used for chaperone assays. The antibodies used in these experiments are anti-SCGN bs-(11744R; Bioss), anti-Hsp70 (MA3-007; Invitrogen), anti-GRP78(C50B12; CST), anti-β-Actin (bs0061R; Bioss), and HRP conjugated secondary antibody (bs0295G; Bioss).
5
Comprehensive Western Blot Procedure for Cell and Tumor Lysates
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The western blot procedure was as outlined in our previous research. In short, both HOS cells and tumors were lysed using chilled RIPA buffer (Beyotime Biotech), combined with a phosphatase and protease inhibitor cocktail (Bioss). After separation by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes (Beyotime Biotech), which were then blocked with 5% non-fat milk for 2 h. The membranes were incubated with the following primary antibodies for a duration exceeding 12 h at a temperature of 4°C: anti-PERK (5683), anti-CHOP (2895), anti-BiP (3177), anti-ATF4 (11815), anti-LC3B (3868), anti-SQSTM1/p62 (88588), anti-Atg5 (12994) anti-cleaved caspase-3 (9664), anti-cleaved PARP (5625), anti-p21 (2947) all at 1:1000, and anti-phospho-eIF2α (3398) at 1:800, all from Cell Signaling Technology. Anti-β-actin (bs-0061R) at 1:5000, anti-phospho-PERK (bs-3330R) at 1:800, and anti-GAPDH (bs-0755R) at 1:5000 from Bioss were also used. Then, the membranes were further incubated with the corresponding HRP-labeled secondary antibodies. Detection was carried out using BiossECL Plus WB Substrate (Bioss), and band intensities were analyzed using Image Lab software.
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