Cells were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. The membranes were blocked with bovine serum albumin or milk for 1 h and probed with use of primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using the GE Amersham Imager 600 system. The levels of proteins were quantified by scanning densitometry of immunoblots (the Fuji Multi Gauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range (Li et al., 2016 (link); Liao et al., 2018 (link); Rezey et al., 2019 (link); Wang et al., 2015 (link), 2018 (link)).
Ge amersham imager 600 system
Manufactured by GE Healthcare
The GE Amersham Imager 600 system is a versatile imaging platform designed for a wide range of applications in life science research. It provides high-quality image capture and analysis capabilities for various types of gel and blot-based assays.
Automatically generated - may contain errors
4 protocols using ge amersham imager 600 system
1
Western Blot Protein Quantification Protocol
2
Protein Quantification in Rat Brain Regions
3
Immunoblot Analysis for Total and Phosphoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was performed according to the methods as previously described21 (link),39 (link)–41 (link). Cells or tissues were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. The membranes were treated with blockers (bovine serum albumin or milk) for 1 h and probed with use of primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using the GE Amersham Imager 600 system. The levels of total protein or phosphoprotein were quantified by scanning densitometry of immunoblots (GE ImageQuant TL Software). The luminescent signals from all immunoblots were within the linear range13 (link),32 (link),39 (link),42 (link),43 (link).
4
Protein Quantification in Rat Brain Regions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rats were anesthetized with pentobarbital sodium (100 mg/kg, MYM Technologies) and rapidly decapitated. The brain was immediately extracted and placed in ice-cold PBS. The prefrontal cortex, hippocampus and cerebellum were dissected and transferred to a 1.5-mL centrifuge tube containing ice-cold RIPA buffer and protease inhibitors (Roche). The tissue was homogenized with an electric homogenizer, then the tube was left on an orbital shaker for 2 h at 4 °C. The cell lysate was centrifuged for 20 min at 12,000 rpm at 4 °C in a microcentrifuge. Supernatants were dissolved in SDS-PAGE sample buffer and boiled for 10 min. Proteins were separated on 8% SDS-PAGE gels. The primary antibodies used were rabbit anti-MeCP2 (1:1000, CST) and mouse anti-β-actin (1:1000, CST). The secondary antibodies used were anti-rabbit and anti-mouse HRP antibodies (1:2000, Sigma-Aldrich). Blots were imaged with a GE Amersham Imager600 system (GE Healthcare, Chicago, IL), and quantified using ImageJ. See Fig. 2A -B in the main text for results.
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!