The assay was based on the original assay of Grajevskaja et al.26 (link) used to discover the E site. The 70S ribosomes used for the assay were prepared as for the minimal in vitro purified translation assay4 . 70S ribosomes (0.2 µM) were incubated in the presence of increasing concentration of EttA-EQ2 (0, 0.2, 0.4, 0.8, 1.0, and 1.2 µM) and deacylated [32P]tRNAPhe (0.4 µM) for 2 min at 4 °C in 0.1 mM ATP, 10 mM Mg(OAc)2, 100 mM NH4Cl, 20 mM Tris-HCl (pH 7.4) in a 20 µl reaction. 5 µl of each reaction were spotted on a nitrocellulose filter (25 mm, 0.45 µm, nitrocellulose, disc filters, Millipore) installed on a Sampling Manifold (Millipore) under constant vacuum. After 3 washes with 2 ml of 20 mM Mg(OAc)2, 100 mM NH4Cl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4), the filters were immersed in scintillation liquid (Ultima Gold, PerkinElmer) and counted on a scintillation counter (Beckman LS6500). 5 µl of each reaction was also counted without being spotted on a nitrocellulose filter or washed, to give the total radioactivity in each reaction. The ratio of radioactivity retained on the filter versus total were calculated and adjusted to the mole of ribosome in each reaction.
Disc filters
Manufactured by Merck Group
Disc filters are a type of filtration equipment used to separate solid particles from liquid or gaseous media. They function by passing the fluid through a series of stacked, perforated discs, which trap the particulate matter while allowing the fluid to pass through.
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Lab products found in correlation
2 protocols using disc filters
1
Monitoring Ribosome E-Site Binding by EttA-EQ2
2
Monitoring Ribosome E-Site Binding by EttA-EQ2
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The assay was based on the original assay of Grajevskaja et al.26 (link) used to discover the E site. The 70S ribosomes used for the assay were prepared as for the minimal in vitro purified translation assay4 . 70S ribosomes (0.2 µM) were incubated in the presence of increasing concentration of EttA-EQ2 (0, 0.2, 0.4, 0.8, 1.0, and 1.2 µM) and deacylated [32P]tRNAPhe (0.4 µM) for 2 min at 4 °C in 0.1 mM ATP, 10 mM Mg(OAc)2, 100 mM NH4Cl, 20 mM Tris-HCl (pH 7.4) in a 20 µl reaction. 5 µl of each reaction were spotted on a nitrocellulose filter (25 mm, 0.45 µm, nitrocellulose, disc filters, Millipore) installed on a Sampling Manifold (Millipore) under constant vacuum. After 3 washes with 2 ml of 20 mM Mg(OAc)2, 100 mM NH4Cl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.4), the filters were immersed in scintillation liquid (Ultima Gold, PerkinElmer) and counted on a scintillation counter (Beckman LS6500). 5 µl of each reaction was also counted without being spotted on a nitrocellulose filter or washed, to give the total radioactivity in each reaction. The ratio of radioactivity retained on the filter versus total were calculated and adjusted to the mole of ribosome in each reaction.
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