Mono q

Manufactured by GE Healthcare

Sourced in United Kingdom, Sweden, India, United States

Mono Q is a laboratory ion exchange chromatography column used for the separation and purification of charged biomolecules. It functions by utilizing a positively charged resin to attract and separate molecules based on their charge characteristics.

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Lab products found in correlation

Superdex 200, by GE Healthcare (12 mentions) Ni nta agarose, by Qiagen (9 mentions) Q sepharose, by GE Healthcare (5 mentions) Bl21 de3, by Thermo Fisher Scientific (4 mentions) Ni2 charged hi trap chelating columns, by GE Healthcare (3 mentions) Ni nta resin, by Qiagen (3 mentions) Superose 6, by GE Healthcare (3 mentions) Ni2 resin, by GE Healthcare (3 mentions) Histrap column, by GE Healthcare (3 mentions) Sp sepharose ff, by GE Healthcare (2 mentions) Akta purifier, by GE Healthcare (2 mentions) Enterokinase, by Evrogen (2 mentions) Superdex 200 pg, by GE Healthcare (2 mentions) Encyclo dna polymerase, by Evrogen (2 mentions) Imac ni2 sepharose, by GE Healthcare (2 mentions) Anti flag m2 affinity gel, by Merck Group (2 mentions) Hiload 16 600 superdex 200, by GE Healthcare (2 mentions) Pierce protease inhibitor tablet, by Thermo Fisher Scientific (2 mentions) Resource q, by GE Healthcare (2 mentions) Hitrap heparin, by GE Healthcare (2 mentions)

Spelling variants of this product

Mono Q column (106 citations) Mono Q 5/50 (8 citations) Mono Q anion exchange chromatography (5 citations) Mono Q-Sepharose fast flow (3 citations) Mono-Q anion exchange (2 citations) Mono-Q GL (2 citations) Mono Q 5/50 GL ion (2 citations) Mono-Q GL column (2 citations) Mono-Q ion-exchange (2 citations) Mono Q 10/100 GL anion exchange chromatography (1 citations)

81 protocols using mono q

Purification of Ctf4 Protein Complexes

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Cleared lysate containing His-tagged full-length Ctf4 or Ctf4_CTD was incubated with 1 ml Ni-NTA resin (QIAGEN), washed with 20 ml Buffer A (50 mM NaH₂PO₄ pH 8.0, 300 mM NaCl, 20 mM imidazole) and eluted five times each with 1 ml Buffer A containing 250 mM imidazole. The resulting elution was dialysed in 2 L 100 mM NaCl, 20 mM Tris pH 8.0, 1 mM DTT for 2 hours with fresh buffer exchanged after the first hour. The dialysed elution was further purified by Mono Q (GE Healthcare) ion exchange through a 0.1-1 M NaCl gradient in 20 mM Tris pH 8.0, 1 mM DTT over 40 ml with 0.5 ml elutions. Peak fractions from the Mono Q were concentrated and polished via Superdex 200 16/600 HiLoad or 10/300 GL (GE Healthcare) size exclusion chromatography in Buffer B (150 mM NaCl, 20 mM Tris pH 8.0). Peak elutions were pooled and concentrated to 5 mg/ml and stored at −80°C in 2 nmol aliquots.

Simon A.C., Zhou J.C., Perera R.L., van Deursen F., Evrin C., Ivanova M.E., Kilkenny M.L., Renault L., Kjaer S., Matak-Vinković D., Labib K., Costa A, & Pellegrini L. (2014). A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome. Nature, 510(7504), 293-297.

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Purification of Mutant RNAP Holoenzymes

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For experiments in Fig. 1B, [531Ser→Leu]β-RNAP σ⁷⁰ holoenzyme was prepared from E. coli strain XE54 (Tang et al., 1994 (link)) transformed with plasmid pRL706-531L [constructed from plasmid pRL706 (Severinov et al., 1997 (link)) by use of site-directed mutagenesis (QuikChange Site-Directed Mutagenesis Kit; Agilent)], using culture and induction procedures, polyethylenimine precipitation, ammonium sulfate precipitation, immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen), and anion-exchange chromatography on Mono Q (GE Healthcare), as in Niu et al., 1996 (link).
For experiments in Fig. S3C, E. coli RNAP σ⁷⁰ holoenzyme and [565Glu→Asp]β-RNAP σ⁷⁰ holoenzyme were prepared from E. coli strain XE54 (Tang et al., 1994 (link)) transformed with plasmid pRL706 (Severinov et al., 1997 (link)) or pRL706-565D (Zhang et al., 2014 (link)), using the same procedures.
For experiments in Fig. 3D, E. coli RNAP σ⁷⁰ holoenzyme was prepared from E. coli strain XE54 (Tang et al., 1994 (link)) transformed with plasmid pREII-NHα (Niu et al., 1996 (link)), using culture and induction procedures, polyethylenimine precipitation, ammonium sulfate precipitation, immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen), and anion-exchange chromatography on Mono Q (GE Healthcare), as in Degen et al., 2014 (link).

Maffioli S.I., Zhang Y., Degen D., Carzaniga T., Gatto G.D., Serina S., Monciardini P., Mazzetti C., Guglierame P., Candiani G., Chiriac A.I., Facchetti G., Kaltofen P., Sahl H.G., Dehò G., Donadio S, & Ebright R.H. (2017). Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase. Cell, 169(7), 1240-1248.e23.

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Purification of T. thermophilus RNAP

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T. thermophilus RNAP core enzyme was prepared was prepared from T. thermophilus strain H8 (DSM 579; DSMZ), using polyethylenimine precipitation, ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose FF (GE Healthcare), anion-excahnge chromatography on Mono Q (GE Healthcare), and cation-exchange chromatography on Mono S (GE Healthcare); T. thermophilus σ^A was prepared from E. coli strain BL21(DE3) transformed with pET28a-Tt-σ^A, using immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen) and anion-exchange chromatography on Mono Q (GE Healthcare); T. thermophilus RNAP core enzyme and T. thermophilus σ^A were combined to yield T. thermophilus RNAP σ^A holoenzyme; and T. thermophilus RNAP σ^A holoenzyme was further purified using size-exclusion chromatography on Superdex 200 (GE Healthcare), as in Zhang et al., 2014 (link).

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Purification of Ctf4 Protein Complexes

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Fluorescent Labeling of A24C PepQ

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A24C PepQ was labelled using either 5-iodoacetamidofluorescein (fluorescein, F), 5-(2-acetamidoethyl) aminonaphthalene 1-sulfonate (EDANS, ED), tetramethylrhodamine-5-iodoacetamide dihydroiodide (tetramethylrhoadmine, TMR), or Oregon Green 488 iodoacetamide (Oregon Green, OG). All dyes were obtained from Invitrogen (Molecular Probes). PepQ (∼10 mg ml⁻¹ in 50 mM Tris, pH 8, 100 mM KCl, 1 mM MgCl₂) was reduced with 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) TCEP and labelled with a 10-fold excess of reactive dye, added in 1 addition for 3 h at 23°C. The reaction was quenched by adding glutathione (5 mM), and the labelled PepQ was first separated from unbound dye by gel filtration (PD-10 column, Pharmacia), followed by re-purification of the labelled protein with high-resolution ion exchange chromatography (MonoQ, GE). The extent of labelling was determined by protein quantification by the Bradford assay (Bio-Rad) and dye quantification under denaturing conditions using known dye molar extinction coefficients37 (link)63 (link). Unique labelling of a single cysteine was verified by both denaturing anion exchange chromatography (MonoQ, GE) in 8 M urea buffer and by detection of a single major and fluorescent tryptic peptide upon separation by C8 reverse-phase chromatography63 (link).

Weaver J., Jiang M., Roth A., Puchalla J., Zhang J, & Rye H.S. (2017). GroEL actively stimulates folding of the endogenous substrate protein PepQ. Nature Communications, 8, 15934.

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Purification of Thermus thermophilus RNAP

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T. thermophilus RNAP core enzyme was prepared from T. thermophilus strain H8 (DSM 579; DSMZ), using cell lysis, polyethylenimine precipitation, ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose FF (GE Healthcare), anion-exchange chromatography on Mono Q 10/100 GL (GE Healthcare), and cation-exchange chromatography on Mono S HR (GE Healthcare), as in Zhang et al., 2012 (link) and Maffioli et al., 2017 (link).
T. thermophilus σ^A was prepared by co-expression of a gene for N-terminally hexahistidine-tagged T. thermophilus σ^A in E. coli, followed by cell lysis, immobilized-metal-ion affinity chromatography on Ni-NTA agarose (Qiagen) and anion-exchange chromatography on Mono Q (GE Healthcare), as in Zhang et al., 2012 (link).
T. thermophilus RNAP σ^A holoenzyme was prepared by combining T. thermophilus RNAP core enzyme and T. thermophilus σ^A, followed by size-exclusion chromatography on Superdex 200 (GE Healthcare), as in Zhang et al., 2014 (link).

Lin W., Das K., Degen D., Mazumder A., Duchi D., Wang D., Ebright Y.W., Ebright R.Y., Sineva E., Gigliotti M., Srivastava A., Mandal S., Jiang Y., Liu Y., Yin R., Zhang Z., Eng E.T., Thomas D., Donadio S., Zhang H., Zhang C., Kapanidis A.N, & Ebright R.H. (2018). STRUCTURAL BASIS OF TRANSCRIPTION INHIBITION BY FIDAXOMICIN (LIPIARMYCIN A3). Molecular cell, 70(1), 60-71.e15.

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Recombinant Protein Purification and Activation

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InpA was produced as a recombinant protein in Escherichia coli and purified as previously described [12 (link), 13 (link)] by affinity chromatography on Fast Flow Ni-NTA (Ni²⁺-nitrilotriacetate)–Sepharose (Qiagen) followed by anion exchange chromatography (MonoQ, GE Healthcare). The active concentration of InpA was determined by active-site titration using an appropriate dilution of a standardized 1 mM aqueous solution of the protease inhibitor N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (E-64) (Sigma-Aldrich; product E3132) needed for total inactivation of the proteinase. Residual enzyme activity was determined by measurement of fluorescence (λ_ex = 380 nm and λ_em = 460 nm) of AMC (7-amino-4-methylcoumarin) released from t-butoxycarbonyl-Val-Leu-Lys-AMC as described previously [13 (link)]. Immediately before use, InpA was activated by incubation for 15 min with 3 mM DTT in 0.1 M Tris–HCl, 0.14 M NaCl, pH 7.5 [50 ]. When used for protein breakdown assays under non-reducing conditions, the above buffer was exchanged for that without reducing agent by ultrafiltration using 10 kDa cut-off Microcons (Amicon).

Byrne D.P., Manandhar S.P., Potempa J, & Smalley J.W. (2015). Breakdown of albumin and haemalbumin by the cysteine protease interpain A, an albuminase of Prevotella intermedia. BMC Microbiology, 15, 185.

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Purification and Application of Trypanosomal Substrate

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TS was expressed in Escherichia coli BL21 and purified to homogeneity by immobilized metal affinity chromatography through Ni²-charged Hi-Trap chelating columns (GE Healthcare) and ion-exchange chromatography (Mono Q; GE Healthcare) as described previously, followed by passage through a polymyxin column (Pierce) for endotoxin depletion (Leguizamon et al., 1999 (link)). As described above (Section Timeline of HIV/Trypanosoma cruzi Co-infection Assays), astrocytes were exposed to 1 μg/ml of TS (active, or inactive isoform) according the Tc/HIV timeline (Figure 2A) replacing trypomastigotes.

Urquiza J.M., Burgos J.M., Ojeda D.S., Pascuale C.A., Leguizamón M.S, & Quarleri J.F. (2017). Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi. Frontiers in Cellular and Infection Microbiology, 7, 345.

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Recombinant XPA Protein Purification

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Human XPA was cloned into pET32a vector (Novagen) with a cleavable TRX-6XHis-S tag at the N terminus and expressed in Rosetta2 cells (Invitrogen). Protein expression was induced by the addition of 0.5 mM IPTG and 50 μM ZnCl₂ at 15°C for 16 hr. The cells were lysed by EmulsiFlex-C5. XPA was purified using HisTrap columns (GE Healthcare) before and after removing the affinity tag by PreScission Protease. The tag-free XPA was further purified with Mono Q and Superdex 200 columns (GE Healthcare). XPA peak fractions in 20 mM Tris (pH 7.4), 150 mM NaCl, 25 μM ZnCl₂, 1mM DTT were pooled and concentrated to 10 mg/ml for storage at −80°C. For activity assays, XPA was thawed and purified over a Superdex 75 column (GE Healthcare) in 25 mM Tris (pH 7.5), 150 mM NaCl, 2 mM DTT, and 5% glycerol.

Li C.L., Golebiowski F.M., Onishi Y., Samara N.L., Sugasawa K, & Yang W. (2015). Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair. Molecular cell, 59(6), 1025-1034.

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Purification and Biotinylation of FmlH Lectin

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FmlH protein used in crystallographic studies was expressed and purified as previously described. Briefly, protein was expressed in the periplasm of E. coli C600 cells containing pTRC99a encoding the first 182 amino acids of the CFT073 FmlH protein (corresponding to the signal sequence and lectin domain) and a C-terminal 6x-his tag. Periplasmic isolates were prepared as previously described and were washed over a cobalt affinity column (GoldBio) and eluted in 20 mM Tris 8.0 + 250 mM imidazole. Fractions containing protein of the expected molecular weight were then diluted 5-fold in 20 mM Tris 8.0 to a final concentration of 50 mM Imidazole, washed over an anion exchange column (GE Healthcare Mono Q) with 20 mM Tris 8.0, and eluted in 20 mM Tris 8.0 + 250 mM NaCl. Resulting fractions were pooled and dialyzed in 1 mM HEPES pH 7.5 + 50 mM NaCl and concentrated as needed for further study. Protein used in ELISA assays was biotinylated using anNHS-PEG4-Biotin and Biotinylation Kits (ThermoFisher).

Maddirala A.R., Klein R., Pinkner J.S., Kalas V., Hultgren S.J, & Janetka J.W. (2019). Biphenyl Gal and GalNAc FmlH Lectin Antagonists of Uropathogenic E. coli (UPEC): Optimization through Iterative Rational Drug Design. Journal of medicinal chemistry, 62(2), 467-479.

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