Samples were mixed with 4x LDS loading dye (Thermo) containing 200 mM DTT and analyzed on Bolt Bis-Tris Plus gels (Thermo) according to manufacturer’s instructions. Gels were either stained with GelCode™ Blue Protein Stain (Fisher Scientific) or prepared for autoradiography by incubation in a 10% polyethyleneglycol 8000 (Sigma) solution for 30 minutes and gel drying at 70°C for 45 minutes using a Hoefer gel drier, Savant condenser unit and an TRIVAC pump (Leybolt). Gels were exposed to phosphor screens for 2 hour to 7 days and developed with a Molecular Dynamics Storm 850 imager. Gel images were examined using ImageJ opensource software. For quantitation, a standard curve was created by dotting known amounts of radioactivity (5 × 10−1 to 5 × 10−5 μCi) on nitrocellulose and subjecting it phosphorimaging. Gel band and standard signal volumes were determined, the background was subtracted, and relative or absolute quantities were determined in Microsoft Excel after linear curve fits of a standard volumes (log (volume) vs log (μCi radioactivity)) that yielded an r2 > 0.99.
Storm 850 imager
Manufactured by GE Healthcare
The Storm 850 imager is a versatile laboratory instrument designed for high-performance imaging and quantification of various molecular targets. It utilizes advanced fluorescence detection technology to capture and analyze images from a wide range of sample types, including gels, blots, and microplates.
Automatically generated - may contain errors
2 protocols using storm 850 imager
1
SDS-PAGE Protein Analysis Protocol
2
Quantitative Protein Analysis by Gel Electrophoresis
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Samples were mixed with 4x LDS loading dye (Thermo) containing 200 mM DTT and analyzed on Bolt Bis-Tris Plus gels (Thermo) according to manufacturer’s instructions. Gels were either stained with GelCodeTM Blue Protein Stain (Fisher Scientific) or prepared for autoradiography by incubation in a 10% polyethyleneglycol 8000 (Sigma) solution for 30 min and gel drying at 70 °C for 45 min using a Hoefer gel drier, Savant condenser unit and an TRIVAC pump (Leybolt). Gels were exposed to phosphor screens for 2 hour to 7 days and developed with a Molecular Dynamics Storm 850 imager. Gel images were examined using ImageJ opensource software. For quantitation, a standard curve was created by dotting known amounts of radioactivity (5 × 10−1 to 5 × 10−5 μCi) on nitrocellulose and subjecting it phosphorimaging. Gel band and standard signal volumes were determined, the background was subtracted, and relative or absolute quantities were determined in Microsoft Excel after linear curve fits of a standard volumes (log (volume) vs log (μCi radioactivity)) that yielded an r2 > 0.99.
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