The kidney tissue was cut into small fragments with scissors, and RIPA lysis (P0013, Beyotime Biotechnology) was used to lyse the tissue on ice, after which the tissue was ground with a grinder to make it fully lysed. The supernatant was obtained after high-speed refrigeration centrifugation, and the protein concentration was determined by a BCA kit (CW0130S, CWBIO, Beijing, China). Electrophoresis was carried out through 8–15% SDS-PAGE, and the sample was transferred to PVDF membranes, then blocked at room temperature for 1 h, and subsequently incubated with the primary antibody at 4 ℃ in incubators overnight. The membranes were washed with PBS and then incubated in the secondary antibody at room temperature for 1 h. The membranes were examined using a chemiluminescence system (Beyotime Biotechnology). The following primary antibodies were used: Nrf2 (PA5-27882), HO-1 (PA5-77833), Bcl-2 (PA5-27094), Bax (MA5-14003), and GAPDH (MA5-15738-D680, Thermo Fisher Scientific).
Chemiluminescence system
Manufactured by Beyotime
Sourced in China
The Chemiluminescence system is a laboratory instrument designed to detect and measure light-emitting chemical reactions. It functions by analyzing the emission of light during specific chemical processes, allowing for the quantification and characterization of various analytes.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Thermo Fisher Scientific (1 mentions)
Ripa lysis, by Beyotime (1 mentions)
Cw0130, by CWBIO (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Chemiluminescence kit, by Beyotime (1 mentions)
β actin, by Immunoway (1 mentions)
Traf6, by Immunoway (1 mentions)
Bca assay reagent, by Beyotime (1 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Bca reagent kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Osterix, by Abcam (1 mentions)
6 protocols using chemiluminescence system
1
Protein Expression Analysis of Kidney Tissue
2
Autophagy Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT22 cells or brain tissue were lysed and the protein concentration was determined with a bicinchoninic acid kit (Beyotime). The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA). The membranes were then incubated in blocking solution (Cat# SW3015BSA; Solarbio, Beijing, China), followed by incubation at 4°C overnight with antibodies for anti-mouse ATG7 (Cat# ab133528; 1:5000; Abcam, Cambridge, UK), anti-mouse Beclin1 (Cat# ab210498; 1:1000; Abcam), anti-mouse light chain 3 (LC3) (Cat# ab128025; 1:500; Abcam), or anti-mouse β-actin (Cat# ab227387; 1:5000; Abcam). Next, the membranes were incubated with goat anti-rabbit IgG secondary antibodies (Cat# ab97048; 1:5000; Abcam) for 3–4 hours at room temperature. The proteins were visualized with a chemiluminescence system (Beyotime) and imaged using the ECL Plus Western Blotting Substrate (Thermo Fisher). The quantitative analysis of the blots was conducted with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues were lysed in ice-cold RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing protease inhibitors and phosphatase inhibitors and then centrifuged at 12000 g for 15 min at 4°C and collected the supernatants carefully. Protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, China). Equivalent amounts of protein were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked in 5% (w/v) nonfat milk powder in TBST (Tris-buffered saline with 0.1% Tween) for 1 h at room temperature. The membranes were incubated with rabbit primary antibodies for TLR4, NF-κB, MYD88, TRAF6, P38, p-P38, JNK1/2, p-JNK1/2, and β-actin from ImmunoWay Biotechnology Company (Plano, TX, USA), respectively, diluted in blocking buffer at 4°C overnight. After washing with TBST for 3 times, the membranes were incubated with peroxidase-conjugated anti-rabbit secondary antibody (1 : 5000, Abcam USA) for 1 h at room temperature and then washed with TBST for 3 times. The immunoblots were developed using a chemiluminescence kit (Beyotime Biotechnology, China) and visualized by a chemiluminescence system (Tanon 5200). The band intensity was quantified using ImageJ, normalized to β-actin.
4
Western Blot Analysis of Notch Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues and cells were homogenized in cold RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 μg/mL PMSF, 1.0 mM sodium orthovanadate, and 1× mammalian protease inhibitor cocktail; Sigma-Aldrich, Shanghai, China). Protein was quantified by the Bradford method using the BCA assay reagent (Beyotime, Shanghai, China). Equal amounts of protein were loaded into SDS-polyacrylamide gels and transferred onto PVDF-Plus membranes. After blocking in 5% fat-free milk for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: NICD2 (ab-52302, Abcam, UK), Hes5 (sc-25395, Santa Cruz, Dallas, CA, USA), and GAPDH (sc-32233, Santa-Cruz, Dallas, CA, USA). The primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG, or goat anti mouse IgG secondary antibody for 1 h at room temperature and detected by the use of a chemiluminescence system (Beyotime, Shanghai, China) and imaging system (Kodak Gel Logic 4000R Imaging System, Carestream, Rochester, NY, USA). A semiquantitative densitometry analysis of the bands were performed using the Kodak Gel Logic 4000R Imaging System (Carestream, Rochester, NY, USA). Protein expression was normalized to the same sample’s expression of GAPDH.
5
Western Blot Analysis of PLCE1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After removing the culture supernatants, the cells were lysed using radioimmunoprecipitation assay buffer (Beyotime). Total protein was harvested and quantified using a BCA Reagent Kit (Beyotime). In brief, 30 μg of protein was separated by electrophoresis on a 10% sodium dodecyl sulphate‐polyacrylamide gel. Partitioned proteins were transferred onto polyvinylidene fluoride membranes, which were blocked in 5% non‐fat milk and then incubated with primary antibodies against PLCE1 (1:500, Biorbyt, orb335375) and β‐actin (1:5000, ProteinTech, 66009‐1‐lg) at 4℃ overnight. Horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse IgG antibody was used as the secondary antibody. Bands were detected using a chemiluminescence system (Beyotime).
6
Western Blot Analysis of Osteogenic Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins were extracted from cells or tissues samples using RIPA lysis buffer (cat. no. P0013E; Beyotime Institute of Biotechnology). The protein concentration was detected using the bicinchoninic acid assay kit (cat. no. BCA1-1KT; Sigma-Aldrich; Merck KGaA). Equal amount of proteins (30 µg per lane) was separated by 12% SDS-PAGE for 40 min. The protein was transferred to a PVDF membrane (EMD Millipore). The membrane was blocked with 5% skimmed milk for 1 h at room temperature. The membrane was washed three times with 1X PBS-0.1% Tween-20 (PBST). The membranes were then incubated with primary antibodies: Runx2 (1:1,000; cat. no. 12556; Cell Signaling Technology, Inc.), ALP (1:1,000; cat. no. sc-365765; Santa Cruz Biotechnology, Inc.), OC (1:1,000; cat. no. ab93876; Abcam), Osterix (1:1,000; cat. no. sc-393060; Santa Cruz Biotechnology, Inc.), and β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc.) overnight at 4°C. Subsequently, the protein was incubated with a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc.) overnight at 4°C. Finally, protein blots were visualized and analyzed using a chemiluminescence system (Beyotime Institute of Biotechnology). β-actin was used as an internal control.
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!