E. coli ribosomes (New England Biolabs) were incubated in labeling buffer (50 mM HEPES pH 8.2, 100 mM KCl, 10 mM magnesium acetate) with a molar excess of Alexa Fluor 488 NHS Ester (ThermoFisher Scientific) for 90 min at room temperature. Free dye was removed by washing with labeling buffer in centrifugal filter devices with a molecular weight cut-off of 100 kDa. Each ribosome contained approximately eleven Alexa Fluor 488 labels.
E coli ribosome
Manufactured by New England Biolabs
E. coli ribosomes are cellular organelles responsible for protein synthesis in Escherichia coli bacteria. They are composed of a small and a large subunit and serve as the site of translation, where genetic information is converted into functional proteins.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 nhs ester, by Thermo Fisher Scientific (1 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Purexpress δ ribosome kit, by New England Biolabs (1 mentions)
E coli rna polymerase holoenzyme, by New England Biolabs (1 mentions)
4 protocols using e coli ribosome
1
Labeling E. coli Ribosomes with Alexa Fluor 488
2
Cell-free Protein Expression Assay
3
Purification of Thermoplasma and Lactococcus Complexes
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Thermoplasma acidophilum 20S proteasome was expressed and purified from Escherichia coli BL21(DE3) strain by following previously reported protocols19 (link). Briefly, we transformed the plasmids into Escherichia coli competent cells, which contained α and β subunits of 20S proteasome, with the N terminal of β subunit tagged by six histidines. After induced expression, Escherichia coli cells were sonicated and the cell lysates were then applied onto affinity nickel column to get purified 20S proteasome sample.
Ll.LtrB RNP were purified from Lactococcus lactis strain IL1403 according to the published methods17 (link). We firstly constructed a plasmid containing Ll.LtrB intron with the ORF region in its DIV depleted, its encoded protein LtrA and a chitin binding domain (CBD), which was then transformed into Lactococcus lactis strain IL1403 and allowed for induction. The cell lysates were loaded onto affinity chitin column, followed by dithiothreitol elution and ultracentrifugation to get purified Ll.LtrB RNP sample.
E. Coli ribosome was purchased from NEB company (P0763S).
Ll.LtrB RNP were purified from Lactococcus lactis strain IL1403 according to the published methods17 (link). We firstly constructed a plasmid containing Ll.LtrB intron with the ORF region in its DIV depleted, its encoded protein LtrA and a chitin binding domain (CBD), which was then transformed into Lactococcus lactis strain IL1403 and allowed for induction. The cell lysates were loaded onto affinity chitin column, followed by dithiothreitol elution and ultracentrifugation to get purified Ll.LtrB RNP sample.
E. Coli ribosome was purchased from NEB company (P0763S).
4
Ribosome Crosslinking with DSAU
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33.3 mg/mL (13.3 µM) E. coli ribosome was purchased from New England Biolabs, cat. no. P0763S20. 20 µL of the commercial solution were diluted to 200 µL with 20 mM HEPES (pH 7.6), 10 mM MgAc2, 30 mM KCl, and 2 mM TCEP. Four 49-µL aliquots were pipetted into four vials. DSAU was purchased from CF Plus Chemicals, cat. no. PCL006_0050. DSAU was dissolved in neat ACN to a final concentration of 5 mM by sonicating for 5 min at room temperature. 1 µL of DSAU stock solution was added to each 49 µL aliquots of 1.33 µM ribosome. The mixtures were incubated for 60 min at room temperature. 1 µL of 1 M ammonium bicarbonate was added and incubated for 15 min at room temperature for quenching the cross-linking reactions.
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