Leader sequence RNA was 5′ labeled with Cy5 (Dharmacon Horizon Inc.). Reactions were incubated at RT for 30 min in buffer containing 20mM HEPES pH 7.8, 30mM KCl, 5mM CaCl2, 30mM NaCl, 0.01% Tween-20, 0.5 TCEP, 0.2 mg/ml BSA (Sigma-Aldrich) and the indicated amount of purified nsp1 protein or 5 μM of the Kaposi’s sarcoma associated herpesvirus RNA binding protein ORF37 as a positive control. Reaction volumes were kept at 20 μL and stopped with 3 μL 7x EMSA loading dye (70mM HEPES pH 8.0, 420mM KCl, 35% glycerol). Reactions were resolved by 8% native PAGE, and gels were imaged on a Typhoon 5 multivariable imager (Cytiva Amersham) and quantified using GelQuant software package (Molecular Dynamics).
Gelquant software package
Manufactured by GE Healthcare
GelQuant is a software package developed by GE Healthcare to analyze and quantify data from gel electrophoresis experiments. The software provides automated tools for image capture, band detection, and data analysis to support research in various fields, including molecular biology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using gelquant software package
1
SARS-CoV-2 nsp1 Binding Assay
2
EMSA Assay for RNA-Protein Interactions
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RNA probes used in EMSA experiments were radiolabeled using the protocol described for ribonuclease activity assays. Reactions were incubated at RT for 30 min in buffer containing 20 mM HEPES pH 8.0, 30 mM KCl, 5 mM CaCl2, 0.01% Tween-20, 0.5 TCEP, 0.2 mg/ml BSA (Sigma-Aldrich), 40 μg/ml of yeast tRNA (Ambion Thermo Fisher), and the indicated amount of purified SOX protein. Calcium Chloride was used in these binding assays to prevent substrate processing and stabilize RNA–protein interactions. Reactions volumes were kept at 10 μl and stopped with 3 μl 7× EMSA loading dye (70 mM HEPES pH 8.0, 420 mM KCl, 35% glycerol). Reactions were resolved by 8% native PAGE, and gels were imaged on a Typhoon multivariable imager (GE Healthcare) and quantified using GelQuant software package (Molecular Dynamics).
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