The experiment was performed as previously described (42 (link)) with some modifications. M. tuberculosis tRNAAsn (final concentration, 2.5 μM) was radiolabeled at 37°C for 30 min with 1 μM E. coli tRNA nucleotidyltransferase and [α-32P]ATP (final activity, 2 μCi/μl) (PerkinElmer; catalog no. BLU003X250UC) in 50 mM Tris-HCl, pH 8.0, 20 mM MgCl2, 5 mM dithiothreitol (DTT), and 50 μM sodium pyrophosphate (NaPPi). After phenol-chloroform (pH < 5.0) extraction, free [α-32P]ATP was removed from the sample via a MicroSpin G-25 column (Cytiva; catalog no. 27-5325-01), and then the tRNA was isopropanol precipitated at −20°C for 2 h. 32P-labeled tRNAAsn (0.4 μM) was diluted with 12.5 μM unlabeled tRNAAsn, and this diluted mixture was used for aminoacylation assays as described above using unlabeled aspartate. The 32P-labeled Asp-tRNAAsn pellet was dissolved in ddH2O to 80 μM and stored in aliquots at −20°C until needed.
Blu003x250uc
Manufactured by PerkinElmer
The BLU003X250UC is a laboratory equipment product manufactured by PerkinElmer. It is designed for use in various scientific and research applications. The core function of this product is to provide a specific set of capabilities to researchers and scientists, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
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3 protocols using blu003x250uc
1
Radiolabeling and Aminoacylation of tRNA Asn
2
Radioactive ATP Uptake Assay for E. coli
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The 100 μL induced E. coli cells (OD600 = 5) were centrifuged and resuspended in 100 μL potassium phosphate buffer containing 25 nM radioactively labeled [α-32P] ATP (1:2000) (PerkinElmer, BLU003X250UC) with uninduced cells as controls. The reaction was carried out at 30°C for a specified time and terminated by adding 400 μL cold potassium phosphate buffer. Further, cells were centrifuged and resuspended three times by adding potassium phosphate buffer to remove unimported radioactivity labeled ATP. Finally, cells were resuspended with 50 μL potassium phosphate buffer and 100 μL ULTIMA Gold (PerkinElmer) in 96-well microplates (PerkinElmer) to measure radioactivity by Perkin Elmer MicroBeta TriLux (PerkinElmer) (22 (link), 23 (link)).
3
Radiolabeling of tRNA Asn for Aminoacylation
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The experiment was performed as previously described ( 23) with some modifications. M. tuberculosis tRNA Asn (final concentration 2.5 µM) was radiolabeled at 37 °C for 30 min with 1 µM E. coli tRNA nucleotidyltransferase and [α- 32 P] ATP (final activity 2 µCi/µL) (PerkinElmer, # BLU003X250UC) in 50 mM Tris-HCl pH 8.0, 20 mM MgCl2, 5 mM dithiothreitol (DTT), and 50 µM sodium pyrophosphate (NaPPi). After phenol/chloroform (pH < 5.0) extraction, free [α-32 P] ATP was removed from the sample via a MicroSpin G-25 column (Cytiva, #27-5325-01) and then the tRNA was isopropanol-precipitated at -20 °C for 2 h. 32 P-labeled tRNA Asn (0.4 µM) was diluted with 12.5 µM unlabeled tRNA Asn and this diluted mixture was used for aminoacylation assays as described above using unlabeled aspartate. The 32 P-labeled Asp-tRNA Asn pellet was dissolved in ddH2O to 80 µM and stored in aliquots at -20 °C until needed.
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