The electrophoresis mobility shift assay of recombinant TRBP protein was described previously (41 (link)). Briefly, the recombinant TRBP protein was incubated with 32P-labeled miLuc-1 or miLuc-2 for 30 min on ice in EMSA binding buffer containing 20 mM Tris–HCl [pH8.0], 1.5 mM MgCl2, 50 mM NaCl, 1.5 mM DTT, 100 ng/μl sonicated salmon sperm DNA, 5% glycerol and 0.4 U/ml RNasein (Promega). The samples were electrophoresed on a 9% non-denatured polyacrylamide gel in 0.25× TBE buffer. The visualization was performed using the Fuji imaging plate and Typhoon image analyzer (GE Healthcare).
Fuji imaging plate
Manufactured by GE Healthcare
Sourced in United States
The Fuji imaging plate is a reusable digital radiography device used for medical imaging. It captures and stores X-ray images electronically, which can then be digitally processed and displayed on a computer monitor.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using fuji imaging plate
1
TRBP Binding to miRNA by EMSA
2
Purification and Binding Assay of RIG-I, MDA5, and LGP2
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Purification of the recombinant RIG-I, MDA5, and LGP2 proteins was performed as described previously [8 (link),14 (link)]. EMSAs were performed in binding buffer (20 mM Tris [pH 8.0], 1.5 mM MgCl2, 50 mM NaCl, 1.5 mM DTT, 100 ng/μL sonicated salmon sperm DNA, 5% glycerol, and 0.4 U/mL RNasin [Promega, Madison, WI, USA]). Each purified protein (3 μM) was incubated with 0.5 nM siRNA containing 32P-labeled guide strand for 30 min on ice. The samples were electrophoresed on a 9% polyacrylamide gel in 0.25 × TBE buffer. For quantification, gels were exposed to a Fuji imaging plate and scanned with a Typhoon image analyzer (GE Healthcare, Chicago, IL, USA).
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