Proteins were isolated from cells with RIPA lysis solution (Beyotime, Shanghai, China). Protein concentrations were measured using a microplate reader (Thermo, Waltham, MA, USA). Total proteins were electrophoresed in 10% sodium dodecyl sulphate–polyacrylamide gels. After electrophoresis, the proteins were transferred onto 0.45 μM polyvinylidene fluoride membranes (PVDF, Millipore, Billerica, MA, USA) and then blocked with 5% skim milk for 2 h. Then, the membranes were incubated with the primary antibodies. The PVDF membranes were incubated with secondary antibodies (HRP-labelled antibodies, Boster, Wuhan, China) for 2 h at 37 °C. Protein bands were visualized using ECL substrate (Applygen, Beijing, China) and chemiluminescence. Band intensities were quantified using ImageJ software. All antibodies are shown in Additional file 2 : Table S2.
Ecl substrate
Manufactured by Applygen
Sourced in China
ECL substrate is a chemiluminescent reagent used in Western blot and other immunoassay techniques to detect and quantify proteins. It emits light upon oxidation of the substrate, which can be detected and measured using a luminometer or X-ray film.
Automatically generated - may contain errors
2 protocols using ecl substrate
1
Western Blotting of Cellular Proteins
2
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were lysed and estimated using a BCA protein assay kit (Beyotime, China). Protein lysates were separated and transferred onto the polyvinylidene difluoride (PVDF) membrane. After blocking, the blots were incubated with specific antibody at 4°C overnight, followed by the incubation of secondary antibody (Abcam). The results were visualized using ECL substrate (Applygen, Beijing, China).
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!