(p)ppApp was quantified using anion-exchange chromatography: cell suspension in lysis solvent equivalent to 1.0 OD600 cells were diluted with aqueous solution of 10 mM Tris-HCl pH 8.0 until the content of organic solvent less than 20%. Insoluble material was pelleted at 10,000 g, and the supernatant was applied to a Mono Q 5/50 column (GE Healthcare) after passing through a 0.22-μm syringe filter. Bound metabolites were eluted at 4°C using a linear gradient of buffer A (5 mM Tris-HCl pH 8.0) and buffer B (5 mM Tris-HCl pH 8.0, 1M NaCl), with the percentage of buffer B increasing from 0 to 35% over 17.5 mL. External standards containing equimolar of AMP, ADP, ATP, pApp, ppApp and pppApp was analyzed under the same condition to locate their peaks. Nucleotides were quantified according to their peak areas on the 254-nm chromatogram.
Mono q 5 50 column
Manufactured by GE Healthcare
Sourced in United States
The Mono Q 5/50 column is a strong anion-exchange chromatography column used for the purification and analysis of biomolecules, such as proteins and nucleic acids. It features a 5 mm internal diameter and a 50 mm bed height, providing a compact design suitable for a variety of laboratory applications.
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10 protocols using mono q 5 50 column
1
Quantification of Nucleotides by Anion-Exchange Chromatography
2
Purification and Kinetics of Tas1tox Enzyme
3
Enzymatic Conversion of Ornithine to Putrescine
4
Quantification of Nucleotides by Anion-Exchange Chromatography
5
Production of Recombinant mSAv-3xHis6 Anchor
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Recombinant mSAv-3xHis6 served as anchor unit for the force sensor and was produced as described33 (link). Briefly, biotin binding deficient subunits equipped with a C-terminal His6-tag—“dead”—and functional subunits with C-terminal 3C protease cleaving site followed by Glu6-tag—“alive”—were expressed as inclusion bodies in E. coli BL-21 using the pET expression system (Novagen). Inclusion bodies were dissolved in 6 M guanidine hydrochloride at 10 mg/ml, mixed at a ratio of 3 “dead” to 1 “alive” subunit and refolded in 100 volumes PBS. The salt concentration was reduced to 2 mM by cycles of concentration using a 10 kDa cutoff spin concentrator (Amicon) and dilution with 20 mM Tris pH 7.0. The mixture was loaded onto a MonoQ 5/50 column (GE Healthcare), and after washing the column with 100 mM NaCl in 20 mM Tris pH 7.0 mSAv-3xHis6 was eluted at 20 mM Tris pH 7.0, 240 mM NaCl. Human 3 C protease (Pierce) was added and allowed to cleave the Glu6-tag overnight. The digested protein was subjected to Superdex 200 (GE Healthcare) size exclusion chromatography in PBS to yield to pure anchor unit.
6
Enzymatic Conversion of Ornithine to Putrescine
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SpeC converts ornithine into putrescine and bicarbonate:
L-ornithine + H+ → CO2 + putrescine
Each reaction (200 μL) contained 50 mM HEPES-Na pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 100 nM SpeC and indicated amounts of GTP-Mg or ppGpp-Mg, and the reaction was started by addition of 1 mM ornithine and allowed to proceed at 37 oC in a water bath for 3 minutes. The reaction was stopped by the addition of 100 μL chloroform and vortexing. The aqueous layer was quantitatively recovered, diluted to 1 mL, and was treated with 100 μL 1 M phthalic anhydride in chloroform at 37 oC for 10 minutes with vigorous shaking. Upon phase separation, 500 μL aqueous layer was applied to a Mono Q 5/50 column (GE Healthcare). Derivatives of ornithine and putrescine were eluted at 4 oC using a linear gradient of buffer A (5 mM HEPES-Na pH 7.0) and buffer B (5 mM HEPES-Na pH 7.0, 1M NaCl), with the percentage of buffer B increasing from 15 to 50% within 10.5 mL. Samples of pure ornithine and putrescine were derivatized and analyzed under the same conditions to identify their derivatives in the chromatogram.
L-ornithine + H+ → CO2 + putrescine
Each reaction (200 μL) contained 50 mM HEPES-Na pH 7.4, 150 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 100 nM SpeC and indicated amounts of GTP-Mg or ppGpp-Mg, and the reaction was started by addition of 1 mM ornithine and allowed to proceed at 37 oC in a water bath for 3 minutes. The reaction was stopped by the addition of 100 μL chloroform and vortexing. The aqueous layer was quantitatively recovered, diluted to 1 mL, and was treated with 100 μL 1 M phthalic anhydride in chloroform at 37 oC for 10 minutes with vigorous shaking. Upon phase separation, 500 μL aqueous layer was applied to a Mono Q 5/50 column (GE Healthcare). Derivatives of ornithine and putrescine were eluted at 4 oC using a linear gradient of buffer A (5 mM HEPES-Na pH 7.0) and buffer B (5 mM HEPES-Na pH 7.0, 1M NaCl), with the percentage of buffer B increasing from 15 to 50% within 10.5 mL. Samples of pure ornithine and putrescine were derivatized and analyzed under the same conditions to identify their derivatives in the chromatogram.
7
Purification and Kinetics of Tas1tox Enzyme
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Each reaction (100 μL total volume) contained 20 mM HEPES-Na 7.4, 300 mM NaCl, 10 mM MgCl2, and substrates at indicated concentrations. Tas1tox was diluted to 10X working concentration in the above buffer conditions and added last. Reactions were incubated at 37°C (Tas1tox turnover experiment in Figure 2g ) or 25°C (all other reactions). At the indicated time points, each 50 μL reaction was diluted in 1 mL ice-cold water and then applied to a MonoQ 5/50 column (GE Healthcare). Bound nucleotides were eluted at 4°C using a linear gradient of buffer A (5 mM Tris-HCl pH 8.0) and buffer B (5 mM Tris-HCl pH 8.0, 1M NaCl), with the percentage of buffer B increasing from 0 to 40% over 20 mL.
8
Sialic Acid Labeling and HPLC Separation
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Oligosialic acid with a degree of polymerization of 6 (oligoSia DP6) was obtained from Nacalai Tesque, Japan. The labeling of oligoSia DP6, polySia avDP18, avDP20, avDP22 and hydrolyzed colominic acid with 1,2-diamino-4,5-methylenedioxybenzenewas (DMB) was carried out over at least 48 hours at 4 °C as previously described by Inoue et al.23 (link). Before the run the sample was set to a pH of 8 to improve binding of the Sia to the column. HPLC retention time was monitored via a Mono Q 5/50 column operated with a flow speed of 0.5 ml/minute (GE Healthcare) in a linear gradient from 0–300 mM NaCl in order to elute and separate the single fractions of polySia avDP18, avDP20 and avDP22.
9
Recombinant Human HPF1 Purification
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To obtain recombinant human HPF1 by expression in E. coli cells, a plasmid was constructed. The HPF1-coding sequence was amplified by PCR using specific primers and total HeLa cDNA. The resulting PCR product was annealed with the linearized pLate31 vector (Thermo Scientific, USA). The amplified DNA plasmid was characterized by the Sanger sequencing method at the SB RAS Genomics Core Facility (ICBFM SB RAS, Novosibirsk, Russia). Next, E. coli Rosetta (DE3) cells were transformed with the pLate31-HPF1 plasmid. The transformed cells were incubated in a Studier autoinduction system in a 1 l of culture. The growth was carried out for 18 h at 18 °C. Further, cells were harvested and lysed, and the HPF1 protein was purified by sequential chromatographies on the Ni-NTA agarose column (GE Healthcare Life Sciences, USA), MonoQ 5/50 column (GE Healthcare Life Sciences, USA), and Superdex 16/600 column (GE Healthcare Life Sciences, USA). The protein concentration was determined spectrophotometrically, using the adsorption coefficient of the protein based on the Expasy Protparam Data.
10
Recombinant HLA-DR Protein Expression
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The extracellular domains of the HLA-DRB1*03:01, 04:01, 15:01 and HLA-DRB5*01:01 beta chains with a basic leucine zipper were cloned into pET28a vector and expressed in E.coli BL21 (DE3) STAR cells (Novagen). Inclusion bodies from E.coli cells were dissolved in 8 M Urea, 50 mM TRIS-HCl buffer pH 8. Proteins were purified using a combination of anion-exchange chromatography on MonoQ 5/50 column (GE Healthcare) followed by sizeexclusion step using Superdex 200 10/300 GL column (GE Healthcare). Protein concentrations were determined based on absorption at 280nm using theoretical extinction coefficients calculated by ProtParam server (https://web.expasy.org/protparam/) from amino acid sequences.
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