Purified preparations of single or triple mutant PKR proteins were incubated at molar ratios of 0.5, 1.0, 2.0, 5.0 and 10 with 50 ng of a duplex 57 nucleotide RNA species derived from the human immunodeficiency virus type I (HIV-1) trans-activation response element (TAR). Protein-RNA complexes were formed in 20 μl of a binding buffer (0.5 × TBE, 0.5% NP40, 0.5% Brij35, 0.5 ng/μl heparin, 0.5 mM argininamide, 10% glycerol) for 1 h at 4 °C, centrifuged at 10,000 g for 1 min, then electrophoretically separated through a native 10% polyacrylamide gel at 200 V, conducted at 4 °C for 20 min in TBE buffer. RNA was stained with a 10,000× dilution of SYBR Gold (ThermoFisher Scientific) in 1 × TBE for 10 min, then visualized with a Typhoon image scanner (GE Healthcare Life Sciences) using excitation with the 488 nM blue laser and emission with the LPB filter (≥510 nM). Images were analysed using ImageQuant 1.2 software (Molecular Dynamics).
Typhoon image scanner
Manufactured by GE Healthcare
The Typhoon image scanner is a high-performance imaging system designed for sensitive fluorescence and luminescence detection. It provides accurate quantification of protein and nucleic acid gels, blots, microarrays, and other samples.
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3 protocols using typhoon image scanner
1
PKR-TAR Interaction Assay
2
Fluorescent PNA Hybridization Assay
3
Brain Perfusion Imaging in Ischemic Rats
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Rats (n 5 10/group) were anesthetized with isoflurane (3%) at 8 d after treatment. 99m Tc-HMPAO (;37 MBq, intravenous) was administered to determine the extent of perfusion defects caused by ischemia and the changes caused by treatment of liposomal angiogenic formulation. Thirty minutes after injection, cervical dislocation was performed. The brains were rapidly excised and fixed with 10% phosphate-buffered formalin for 30 min. Later, the brains were sectioned (2-mm thickness) perpendicular to the long axis of the brain (4 slices) covering the MCA territories. The slices were exposed to imaging plates for 30 min. The images were scanned using a Typhoon image scanner (GE Healthcare) to determine the activity of 99m Tc-HMPAO.
Quantitative image analysis was performed using Multi-Gauge software (Fuji Film). Activity of 99m Tc-HMPAO in the ipsilateral ischemic cortex of the brain (left cortex) versus that in the contralateral cortex (right cortex) was estimated using region-of-interest analysis. Specifically, 99m Tc-HMPAO activity in the ipsilateral cortex was normalized to that in the contralateral cortex. The results were expressed as a ratio.
Quantitative image analysis was performed using Multi-Gauge software (Fuji Film). Activity of 99m Tc-HMPAO in the ipsilateral ischemic cortex of the brain (left cortex) versus that in the contralateral cortex (right cortex) was estimated using region-of-interest analysis. Specifically, 99m Tc-HMPAO activity in the ipsilateral cortex was normalized to that in the contralateral cortex. The results were expressed as a ratio.
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