Proteins were extracted from the cells with RIPA Lysis Buffer I (Sangon Biotech), and the concentration was determined according to standard protocols of the BCA Protein Assay Kit (Sangon Biotech). The total protein (30 μg/well) in the supernatant was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 2 h, the membrane was incubated with primary antibodies against galectin-3 (dilution 1 : 1000; cat. no. ab209344; Abcam, UK), LC3B (dilution 1 : 1000; cat. no. ab192890; Abcam), caspase 3 (dilution 1 : 1000; cat. no. ab32351; Abcam), caspase 9 (dilution 1 : 1000; cat. no. ab32539; Abcam), cytochrome 9 (dilution 1 : 2000; cat. no. ab134909; Abcam), Bax (dilution 1 : 1000; cat. no. ab32503; Abcam), Bcl-2 (dilution 1 : 1500; cat. no. ab32124; Abcam), and GAPDH (dilution 1 : 5000; cat. no. ab8245; Abcam) at 4°C overnight and then incubated with secondary antibodies (dilution 1 : 10,000; cat. no. ab6940; Abcam) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software (National Institutes of Health).
Enhanced chemiluminescence reagent
Manufactured by Sangon
Enhanced chemiluminescence reagent is a laboratory product designed to facilitate chemiluminescence detection. It is used in various applications that involve the analysis of protein expression or other biomolecules through chemiluminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Sangon (3 mentions)
Ripa lysis buffer 1, by Sangon (1 mentions)
Anti sirt1, by ABclonal (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Quantity one 1 d analysis software, by Bio-Rad (1 mentions)
Ab75973, by Abcam (1 mentions)
Ab157208, by Abcam (1 mentions)
Ab155963, by Abcam (1 mentions)
Ab190775, by Abcam (1 mentions)
Ab269514, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Total protein extraction kit, by Sangon (1 mentions)
4 protocols using enhanced chemiluminescence reagent
1
Western Blot Analysis of Apoptosis Markers
2
Investigating Acetylated p53 Regulation in Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cardiomyocytes were transfected or induced with H2O2 as described earlier. Subsequently, protein levels of SIRT1, p53 and acetylated p53 were determined by western blotting. In brief, cells were lysed to isolate total proteins using RIPA lysis buffer (Beyotime Institute of Biotechnology), and total concentrations were determined using the bicinchoninic acid method. Protein samples (50 µg per lane) were subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. Non-specific antigens in the PVDF membranes were blocked using 5% skimmed milk at room temperature for 1 h. Following washing in TBS-0.05% Tween-20, the membranes were incubated with the following primary antibodies (dilution, 1:1,000; all ABclonal Biotech Co., Ltd.) at 4°C overnight: Anti-SIRT1 (cat. no. A19667), anti-p53 (cat. no. A19585), anti-acetyl-p53 (cat. no. A16324) and anti-GAPDH (cat. no. AC001). Following the primary antibody incubation, the membranes were incubated with the secondary antibody (dilution, 1:1,000; cat. no. AS014; ABclonal Biotech Co., Ltd.) at room temperature for 1 h. Band exposure was achieved using enhanced chemiluminescence reagent (Sangon Biotech Co., Ltd.). Protein expression levels were quantified using Quantity One® 1-D Analysis software (version 4.6.8; Bio-Rad Laboratories, Inc.).
3
Protein Expression Analysis in Iron Metabolism
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted, and the concentration was determined according to standard protocols of protein extraction and BCA protein assay kits, respectively (Sangon Biotech, Shanghai, China). The total protein in the supernatants (30 μg/well) was separated by 10% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-hepcidin (1 : 5000; no. ab190775, Abcam, Cambridge, MA, USA), anti-TfR1 (1 : 5000; no. ab269514, Abcam), anti-Ferritin (1 : 5000; no. ab75973, Abcam), anti-DMT1 (1 : 1000; no. ab157208, Abcam), anti-ALK2 (1 : 500; no. ab262699, Abcam), anti-BMP6 (1 : 1000; no. ab155963, Abcam), anti-SMAD1/5/8 (1 : 1000; orb162781, Biorbyt, UK), and anti-GAPDH (1 : 5000; no. ab8245, Abcam) antibodies. After three washes with TBST, the immunoblots were incubated with alkaline phosphatase-labeled goat anti-rabbit antibodies (1 : 1000, Cell Signaling Technology, USA). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Sangon Biotech). The blots were semiquantified by ImageJ software 1.47 (National Institutes of Health, Bethesda, MD, USA).
4
Protein Expression Analysis of Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells (hWJMSCs, hBMSCs, and chondrocytes) and tissues were extracted used the Total Protein Extraction Kit (Sangon Biotech). Next, BCA Protein Assay kits (Sangon Biotech) were used to determine the protein concentration. The total protein was separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to P membrane, and blocked in 5% skimmed milk. Subsequently, the membrane was incubated overnight with exosome-associated primary antibodies (CD63, CD9, and TSP70) as well as the following primary antibodies: ITGB1, TGF-β, p-Smad2/3, Smad2/3, Smad6, COLL-II, and β-actin. The next day, all membranes were washed with PBST three times, and then incubated with the appropriate secondary antibody for 1 h. An enhanced chemiluminescence reagent (Sangon Biotech) was used to visualize the immunoreactive bands. The strength of each band was detected using ImageJ software (National Institutes of Health, USA).
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!