Cells were inoculated and treated as above, and protein concentrations were assessed by bicinchoninic acid (BCA) assay (Beyotime Shanghai, China). The proteins were separated in equal quantities (20 µg) on 10% or 8% gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by electrophoresis (Sigma–Aldrich, St. Louis, MO, USA). Subsequently, western blotting was performed using anti-PGC-1α, p-JNK, JNK or anti-GAPDH antibodies. GAPDH was used as a loading control for cell lysates to normalize PGC-1α and p-JNK levels. Finally, a BeyoECL Plus Ultra-sensitive ECL Chemiluminescence Kit (Beyotime Shanghai, China) and Omega Lum W imaging system BioRad ChemiDoc XRS imaging system (Hercules, CA, USA) were used to observe the immune response bands corresponding to PGC-1α and p-JNK.
Beyoecl plus ultra sensitive ecl chemiluminescence kit
Manufactured by Beyotime
Sourced in China
BeyoECL Plus Ultra-sensitive ECL Chemiluminescence Kit is a reagent used for the detection of target proteins in Western blot analysis. It generates a chemiluminescent signal upon interaction with the target protein, which can be detected and quantified using a suitable imaging system.
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3 protocols using beyoecl plus ultra sensitive ecl chemiluminescence kit
1
Western Blot Analysis of PGC-1α and JNK
2
Western Blot Analysis of Apoptosis Markers
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After lysing using the RIPA Lysis Buffer (Beyotime), samples were separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were processed using a BeyoECL Plus Ultrasensitive ECL Chemiluminescence Kit following the manufacturer's protocol (Beyotime). The following primary antibodies from Abcam (Shanghai, China) were used: anti-Bax (1 : 1000; ab32503), anticleaved caspase-3 (1 : 500; ab32042), anticaspase-3 (1 : 5000; ab32351), and anti-GAPDH (1 : 2500; ab9485). HRP-labeled anti-IgG was used as the secondary antibody. GAPDH is a loading control.
3
Western Blot Analysis of Cell Signaling
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Cells collected by pre-cooled PBS washing were resuspended in cell lysis buffer (Beyotime, China) with phosphatase and protease inhibitors (Beyotime, China). After vortexing and lysing on ice for 10 minutes, protein samples were prepared. Protein concentration was tested using with BCA kit (Beyotime, China). PAGE gels were prepared and loaded with marker and equal amounts of protein samples for separation. Proteins were transferred from gel to PVDF membrane. The membrane was then soaked in 5% skim milk for 60 minutes. After washing with TBST, membrane was incubated with primary antibody at 4°C overnight. Antibodies against Ki-67 were obtained from Huabio (China), and antibodies against IGF1, PARP, Bcl-2, Bax, and cleaved caspase 3 were obtained from ABclonal (China). After washing with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody for 2 hours at room temperature. Protein bands were assessed using the BeyoECL Plus ultra-sensitive ECL chemiluminescence kit (Beyotime, China).
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