Proteins expression was measured by western blotting as described previously (28 (link)). Nuclear and cytoplasmic proteins were extracted from cultured cells using a nuclear or cytoplasmic protein extraction kit (Beyotime Institute of Biotechnology), respectively (Cells were seeded into plates at a density of 3.5 × 104/ml). Protein concentrations were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). Then, cell lysates (10 µg/lane) were separated by SDS-PAGE (10%) and electrophoretically transferred onto a polyvinylidene fluoride membrane. The membrane was then washed with TBS with Tween-20 (TBST) three times, and blocked with 5% non-fat milk in TBST (1% Tween-20) for 2 h at room temperature. The membrane was incubated with Nrf2 (1:1,000), keap1 (1:300), p65 (1:3,000), IκBα (1:1,000), GAPDH (1:1,000) and Lamin B (1:1,000) antibodies overnight at 4°C, and incubated with HRP-coupled secondary antibodies (1:2,000) for 1 h at room temperature. Proteins were detected using enhanced chemiluminescence (Fdbio Science). The protein intensity was measured using Image J software (version 1.48; National Institutes of Health). The relative protein levels were normalized to GAPDH/Lamin B1 protein.
Enhanced chemiluminescence
Manufactured by Fdbio
Sourced in China
Enhanced chemiluminescence is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It relies on a chemical reaction that produces light, which can be measured and used to determine the presence and amount of the target analyte. The core function of this equipment is to facilitate this chemiluminescent detection process.
Automatically generated - may contain errors
Lab products found in correlation
Nuclear or cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by BestBio (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab6721, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab6728, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab202068, by Abcam (1 mentions)
4 protocols using enhanced chemiluminescence
1
Western Blot Analysis of Nuclear and Cytoplasmic Proteins
2
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor (BestBio, Shanghai, China). Protein was loaded onto SDS-PAGE gels, separated through electrophoresis, and transferred to PVDF membranes (Merck KGaA, Darmstadt, Germany). The membranes were incubated with the target primary antibody (PARP, BAX, BCL-2, LC3, P62, PINK1, Parkin and GAPDH) at 4° C overnight and then with the secondary antibody (1:5000, Santa Cruz Biotechnology, TX, USA) at room temperature for 2 h. Immunoblots were visualized using enhanced chemiluminescence (Fdbio Science, Hangzhou, China) and a Tanon-S200 imaging system (Shanghai, China). Bands were quantified and normalized to the loading control.
3
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After drug treatment, total cell lysis of MIHA and AML12 cells was performed using RIPA lysis buffer (Beyotime). The protein concentration of each sample was quantified using a bicinchoninic acid assay. Protein samples (50 μg) were separated on 5% to 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking with 5% bovine serum albumin, membranes were incubated with anti-human/mouse Nrf2 (A0674; Abclonal, Wuhan, China), anti-human/mouse GPX4 (52455l; Cell Signaling Technology), anti-ALOX12 (ER1903-58; Huabio, Hangzhou, China), anti-MCU (ER1803-57; Huabio), anti-MICU1 (ER1803-99; Huabio), anti-Ferritin (ET1610-78, Huabio, Hangzhou, China), or anti-human/mouse β-actin (ab6276; Abcam) antibodies at a dilution of 1:1,000 at 4 °C overnight, respectively. After washing with Tris-buffered saline containing Tween, the membranes were incubated with secondary HRP-conjugated goat anti-rabbit IgG (ab6721; Abcam) or goat anti-mouse IgG (ab6728; Abcam) antibodies at a concentration of 1:1,000 at 25 °C. One hour later, membranes were washed with phosphate buffered saline with Tween-20, and proteins were visualized using enhanced chemiluminescence (FDbio, Hangzhou, China). Protein expression was analyzed according to the gray value using ImageJ software.
4
Caerin 1.1 and 1.9 Mixture-Induced Apoptosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Caerin 1.1 and 1.9 mixture (molar ratio, 1:1) treated HeLa cells were diluted in SDS–PAGE sample buffer before mixed with loading buffer, electrophoresed through an 8% SDS–PAGE gel and then transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in PBS and probed with different monoclonal antibodies (Abcam, ab202068 for caspase 9; Abcam, ab13847 for caspase 3; Abcam, #4249 for PI3K) at a dilution of 1:1000 respectively. Bound antibody was detected by incubation of the membrane with horseradish-peroxidase conjugated goat anti-rabbit/mouse antibody (PTG) at a dilution of 1:1000 and visualized using enhanced chemiluminescence (Fdbio science, Hangzhou, China) and visualized by an image scanner ProteinSimple (Santa Clara, CA, United States).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!