Cortex tissues or RBMEC were lysed with RIPA lysis buffer on ice for 15 min. A BCA protein assay kit was used for examining total protein content. After separating the protein products (50 µg protein/lane) on 10% gels using SDS polyacrylamide gel electrophoresis, they were transferred onto PVDF membranes, blocked with 5% nonfat milk for 2 h at 25˚C. Subsequently, primary antibodies including SIRT1 (1:20,000), BDNF (1:2,000), SYN (1:20,000), MAP-2 (1:20,000), NGF (1:3,000), NT-4 (1:3,000) and Tau-1 (1:1,000), PSD95 (1:1,000) and GAPDH (1:50,000) were added to the membranes for incubation overnight at 4˚C. Then, the membrane was placed in HRP-conjugated secondary antibodies (1:1,000), along with incubation for 1 h at room temperature. The protein bands were developed with an ECL detection Kit and analyzed with fully automatic chemiluminescence imaging system (Tanon 5200Multi; Tanon Science & Technology).
Tanon 5200multi
Manufactured by Shanghai Tanon Science & Technology
Sourced in China
The Tanon 5200Multi is a versatile lab equipment designed for various applications in scientific research and analysis. It is a multi-functional instrument that can perform a range of tasks, including gel electrophoresis, Western blotting, and imaging. The Tanon 5200Multi is engineered to provide consistent and reliable results, making it a valuable tool for researchers and technicians in various fields.
Automatically generated - may contain errors
3 protocols using tanon 5200multi
1
Western Blot Analysis of Neuronal Proteins
2
Immunoblotting Technique for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting assays were performed as previously described [18 (link)]. SDS-PAGE was performed on the extracted proteins, which were then transferred onto PVDF membranes (BIO-RAD, Hercules, CA, USA). The gel bands formed after exposure to appropriate antibodies (primary and secondary) were visualized using the ECL reagent (BIO-RAD) and imaged by Tanon 5200-Multi (Tanon Science & Technology, Shanghai, China). The primary antibodies (against TFEB, GFP, LAMP2, NDRG1, SMPD1, and actin) and the HRP-conjugated secondary antibodies (goat anti-rabbit antibody) were supplied by Proteintech (Wuhan, China). Original uncropped images for immunoblotting are presented in Figure S1 .
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Target cells were incubated with the indicated factors for the designated times and subjected to Western blot analysis. Whole-cell lysates were extracted using Blue Loading Buffer Pack (Cell Signaling Technology, Danvers, MA, USA, 7722S) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF). Fifty micrograms of mouse liver tissue was homogenized and lysed in RIPA buffer supplemented with 1 mmol/L PMSF to extract total protein. Protein concentrations were determined by BCA protein assay kit and all samples were diluted to the same concentration before denaturation. Sixty micrograms of protein was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, transferred onto a nitrocellulose membrane (NC) membrane by wet transfer (200 mA, 90 min), and incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (all primary and secondary antibodies are presented in Supporting Information Table S1 ). The immunoreactive bands were detected by ECL reagent (Vazyme Biotech, Nanjing, China) using a gel imaging system (Tanon 5200 Multi, Tanon Science & Technology, Shanghai, China). The intensity of the bands was analyzed using the Image J software (NIH, 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!