EMSA assays were carried out by incubating RIG-I (or RIG-I construct), fluorophore-tagged dsRNA (3’-DY547, 5’-Biotin) and a 4-fold excess of monovalent streptavidin at 4°C in Buffer A for 60 minutes. An equimolar concentration (25 nM) of RIG-I or the specified RIG-I construct and the fluorophore-tagged dsRNA was used for the EMSAs, unless stated otherwise. 2 mM ATP or 1 mM ATP analog was added to the reaction as specified in the figure legends, 15 minutes before loading the sample on the gel. Loading buffer (10× concentration is 1.5% Ficoll 400 in the Tris-borate buffer, pH 8.0) was added to the samples and run on a 4–16% gradient Native PAGE gel (Invitrogen) at 4°C. The gels were scanned at 532 nm using a Typhoon FLA 9500 laser-based scanner (GE).
Typhoon fla 9500 laser based scanner
Manufactured by GE Healthcare
The Typhoon FLA 9500 is a laser-based scanner designed for high-performance fluorescence and chemiluminescence detection. It is capable of scanning a wide range of sample types, including gels, blots, and microplates, with high sensitivity and resolution.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using typhoon fla 9500 laser based scanner
1
RIG-I Binding to dsRNA Visualization
2
EMSA Assay for RIG-I and LGP2 Binding to dsRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EMSA assays were conducted by incubating RIG-I or LGP2 with fluorophore-tagged dsRNA at 4°C in Buffer A (comprising 50 mM MOPS, pH 7.4, 0.05 mM BSA, 5 mM DTT, 5 mM MgCl2, 0.01% Tween20) for 60 min. The EMSA reactions utilized a range of RIG-I or LGP2 concentrations (as indicated in the legend) at 1.5 to 12-fold molar excess relative to the concentration of fluorophore-tagged biotinylated dsRNA (25 nM). The reactions were supplemented with 2 mM ATP, as specified in the figure legends. Subsequently, loading buffer (comprising 1.5% Ficoll 400 in Tris-borate buffer, pH 8.0) was added to the samples, which were then loaded onto a 4–20% gradient native polyacrylamide gel in TBE buffer (Invitrogen). The gels were scanned at 532 nm using a Typhoon FLA 9500 laser-based scanner (GE) and quantified using ImageQuant software.
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!