Samples extracted from BEAS-2B cells were homogenized in lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China). The concentration of proteins was quantified by virtue of bicinchoninic acid (BCA) protein assay kits (Thermo Fisher Scientific Inc.) according to the operating manual provided by manufacturers. Following the separation with 8% SDS-PAGE, proteins (20 μg per lane) were loaded onto PVDF membranes (BioRad, Hercules, CA). Blocked with 5% BSA, membranes were immunoblotted with primary antibodies against KLF6 (cat. no. 14716-1-AP; 1:1000, Proteintech), SIRT4 (cat. no. 66543-1-Ig; 1:20000, Proteintech), and GADPH (cat. no. 60004-1-Ig; 1:50000, proteintech) at 4°C overnight and horseradish peroxidase-labeled goat anti-rabbit secondary antibody (cat. no. HRP-11449; 1:5000, Proteintech) for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescent (ECL) kit (Bio-Rad Laboratories, Inc, CA, USA), and the relative density of each band was calculated with Image J software (Version 1.49; National Institutes of Health). GADPH served as a control to standardize the results.
Enhanced chemiluminescent ecl kit
Manufactured by Bio-Rad
Sourced in United States
The Enhanced chemiluminescent (ECL) kit is a laboratory equipment product designed for protein detection and analysis. It utilizes a chemiluminescent reaction to enable the visualization of target proteins on Western blots. The kit provides the necessary reagents to facilitate this process.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase labeled goat anti rabbit secondary antibody, by Proteintech (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Np 40 lysis buffer, by Solarbio (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
2 protocols using enhanced chemiluminescent ecl kit
1
Western Blot Analysis of Protein Expression
2
Western Blotting Protocols for Protein Detection
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All cell samples were harvested, and lysed with NP-40 lysis buffer (Solarbio, China) and mixed with 5× sample loading buffer. Next, the proteins were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels with equal amounts and transferred onto polyvinylidene fluoride (PVDF) membranes as described previously (Wang et al., 2019 (link)). The PVDF membranes were blocked with 2.5% skimmed milk and incubated with anti-Nrf2, anti-HO-1 mAb, anti-α-Tubulin mAb (Servicebio, China), or anti-EqHV-8 gD polyclonal antibody. HRP-conjugated goat anti-mouse or goat anti-rabbit IgG was used as the secondary antibody. Then, the protein band signals were detected by ChemiDoc XRS imaging system (Bio-Rad) with an Enhanced Chemiluminescent (ECL) kit (Bio-Rad, USA).
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