Transfer RNA was purified from K12 strain of E. coli using Tri-reagent by differential precipitation with sodium salts as described before.63 (link) Isolated tRNA was reprecipitated with 3M ammonium acetate to replace the sodium ions at the final purification step before dissolving it in water. RNase T1 and bacterial alkaline phosphatase were procured from Worthington Biochemical Corporation. The RNases MC162 (link) and cusativin61 (link) were overexpressed in BL21 and Rosetta™ (DE3) (Novagen) cells, respectively, and purified on a nickel column using His-tag protein purification kit (EMD Millipore) as per the manufacturer’s guidelines. Each batch of ribonuclease protein preparation was evaluated for optimal RNA:protein ratio to ensure appropriate digestion of substrate RNA. In general, optimal digestion of the tRNA mixture or rRNA was carried out by mixing 0.5–1.0 μg of ribonuclease protein for each μg of RNA in the presence of 120 mM ammonium acetate (pH not adjusted) for 90 min at 37 °C for MC1 and 62 °C for cusativin.
His tag protein purification kit
Manufactured by Merck Group
Sourced in United States
The His-tag protein purification kit is a laboratory tool designed to isolate and purify recombinant proteins that have a histidine (His) tag attached. The kit includes all the necessary components, such as affinity resin and buffers, to enable efficient capture and elution of the target protein. This product facilitates the extraction and purification of His-tagged proteins from complex mixtures, which is a common technique in molecular biology and biochemistry research.
Automatically generated - may contain errors
Lab products found in correlation
Rnase t1, by Worthington (2 mentions)
Bacterial alkaline phosphatase, by Worthington (1 mentions)
Yeast trnaphe, by Merck Group (1 mentions)
Escherichia coli trna, by Roche (1 mentions)
Riboflavin, by Thermo Fisher Scientific (1 mentions)
E coli trnatyr, by Merck Group (1 mentions)
Rnase t1, by Roche (1 mentions)
3 protocols using his tag protein purification kit
1
Purification and Enzymatic Digestion of tRNA
2
Customized RNA Sequence Analysis
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A customized RNA (5′-GCAUCAGAAAUACACCCGUAGGGCUUU-GAGA-3′) with all the dinucleotide combinations (42 = 16) and four homopolymer sequences was purchased from Integrated DNA Technology (IDT). Yeast tRNAPhe was obtained from Sigma, and all other chemicals were procured from Thermo Fisher Scientific. RNase T1 was obtained from Worthington Biochemical Corporation. RNase MC1 [11 (link)] and cusativin [12 (link)] were purified on a nickel column using His-tag protein purification kit (EMD Millipore) following their expression from recombinant plasmids in Rosetta TM (DE3) and BL21 strains of Escherichia coli, as described before.
3
Purification of Ribonuclease Cusativin
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Escherichia coli tRNA and RNase T1 were procured from Roche Diagnostics (Indianapolis, IN, USA). E. coli tRNATyr was obtained from Sigma-Aldrich (St. Louis, MO, USA). Riboflavin and all other chemicals were acquired from Fisher Scientific (Fairlawn, NJ, USA) unless otherwise specified. Ribonuclease cusativin was purified from the overexpression strain of E. coli on a nickel column using a His-tag protein purification kit from EMD Millipore (Burlington, MA, USA) as described [51 (link)].
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