Western blots were performed using standard protocols. Cell lysates were prepared using RIPA buffer. Protein samples were subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (Millipore). The membrane was blocked with 5% non-fat milk. The membrane was then incubated with anti-E-Cadherin antibody (1:1000, bs-10009R, Bioss, Beijing, China), anti-Vimentin antibody (1:1000, bs-0756R, Bioss, Beijing, China), anti-TRAF6 antibody (1:1000, bs-1184R, Bioss, Beijing, China), anti-N-Cadherin antibody (1:1000, AF5237, Beyotime, Shanghai, China), anti-RGS2 antibody (1:1000, sc-100761, Santa Cruz), anti-Flag antibody (1:1000, M185-3, MBL, Beijing, China) or anti-αTubulin antibody (1:5000, AF5012, Beyotime, Shanghai, China) at 4 °C overnight, followed by incubation with HRP-anti-rabbit or HRP-anti-mouse secondary antibody at room temperature for 1 h. Signals were detected using an ECL Kit (P0018AS, Beyotime, Shanghai, China) according to the manufacturer’s protocol.
Bs 10009r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-10009R is a piece of lab equipment designed for general laboratory use. It serves as a centrifuge, which is a device used to separate components of a liquid sample based on their density differences. The Bs-10009R operates at a maximum speed of 10,000 rpm and has a maximum capacity of 6 x 50 mL tubes. Its core function is to facilitate the separation and isolation of different biological or chemical components within a liquid sample.
Automatically generated - may contain errors
Lab products found in correlation
Bs 1303r, by Bioss Antibodies (2 mentions)
Sab4200500, by Merck Group (2 mentions)
Bs 1165r, by Bioss Antibodies (2 mentions)
Sc 100761, by Santa Cruz Biotechnology (1 mentions)
Bs 0756r, by Bioss Antibodies (1 mentions)
Af5012, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Sc 373800, by Santa Cruz Biotechnology (1 mentions)
P gsk3β ser9, by Santa Cruz Biotechnology (1 mentions)
Protein extraction kit, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by Cytiva (1 mentions)
Bs 2188r, by Bioss Antibodies (1 mentions)
Bs 0295m hrp, by Bioss Antibodies (1 mentions)
Sodium dodecyl sulfonate polyacrylamide gel, by Solarbio (1 mentions)
Bs 0623r, by Bioss Antibodies (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Tiangen Biotech (1 mentions)
Ecl reagent, by Vazyme (1 mentions)
Dm4000b, by Leica (1 mentions)
4 protocols using bs 10009r
1
Western Blot Analysis of EMT Markers
2
Protein Expression Analysis in Kidney Tissue
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Total protein from the kidney tissue and NRK-52E cells was obtained with a protein extraction kit (Solarbio, Beijing, China) as directed by the manufacturer. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate proteins (50 μg/well). When bromophenol blue ran to the bottom of the gel, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. Blocking was carried out with 5% skim milk. The membranes were then washed with TBST three times for 10 min and subjected to incubation with the following antibodies overnight: β-catenin (1:500, bs-1165R, Bioss), Sfrp1 (1:500, bs-1303R, Bioss), p-GSK3βser9 (1:500, sc-373800, Santa Cruz), E-cadherin (1:500, bs-10009R, Bioss), and α-SMA (1:500, 55135-1-AP, Proteintech), collagen IV (col-IV) (1:1000, SAB4200500, Sigma), β-actin (1:4000, Lot:181620, Pumei), and GSK3β (1:500, sc-8257, Santa Cruz). The next day, the membranes underwent three TBST washes of 10 min. Secondary antibodies diluted with 1% skim milk were added to the membranes for 1 h at room temperature. Band intensities were assessed by Image Lab software.
3
Protein Quantification and Western Blot Analysis
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Total protein in GC cells was extracted utilizing RIPA buffer (Beyotime) and determined utilizing a BCA protein assay kit (Tiangen, Beijing, China). Then the equal amount of protein (30 μg) was separated by sodium dodecyl sulfonate–polyacrylamide gel (Solarbio) and blotted onto polyvinylidenedifluoride membranes (Amersham Biosciences, Chicago, IL, USA). After blocking in 5% defatted milk for 1 h, the membranes were cultivated overnight with primary antibodies against CyclinD1 (1:2,000; bs-0623R; Bioss, Beijing, China), E-cadherin (1:2,000; bs-10009R; Bioss), N-cadherin (1:2,000; bs-1172R; Bioss), or GAPDH (1:5,000; bs-2188R; Bioss) at 4°C followed by interaction with HRP-conjugated secondary antibody (1:5,000; bs-0295M-HRP; Bioss) for 1.5 h at room temperature. The protein bands were visualized with an ECL reagent (Vazyme, Nanjing, China) and analyzed via ImageJ v1.8.0 (National Institutes of Health).
4
Immunohistochemistry and Immunofluorescence Analysis
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For immunohistochemistry, a two-step immunoassay kit (ZSBIO, Beijing, China) was used for streptavidin-peroxidase (SP) detection. For immunofluorescence, NRK-52E cells were grown in 12-well plates, stimulated with normal or high glucose, and transfected with miR-27a inhibitor, miR-27a inhibitor negative control, si-sfrp1, and combined miR-27a inhibitor and si-sfrp1. After treatment for 48 h, the cells underwent fixation (4% formalin at room temperature, 20 min), permeabilization (0.1-0.3% Triton X-100, 10 min), and blocking (3% BSA at 37°C, 30 min). Next, sfrp1 antibody (1:50; bs-1303R, Bioss), β-catenin antibody (1:50; bs-1165R, Bioss), E-cadherin antibody (1:50; bs-10009R, Bioss), α-SMA antibody (1:50; 55135-1-ap, Proteintech), and col-IV antibody (1:50; SAB4200500, Sigma) were added for overnight incubation at 4°C. The cells were then rinsed with ice-cold PBS, and the corresponding fluorescent secondary antibodies were added for incubation (37°C, 1 h). DAPI staining was performed for 5 min, and the cells were observed under an immunofluorescence microscope (Leica, DM4000B, Germany).
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