Hiload 16 60 superdex 200 size exclusion column

Manufactured by GE Healthcare

The HiLoad 16/60 Superdex 200 size exclusion column is a chromatography column designed for the separation and purification of proteins and other macromolecules. It is a prepacked column with a bed volume of 120 mL, using Superdex 200 resin. The column is intended for use in fast protein liquid chromatography (FPLC) systems and can handle sample volumes up to 2.4 mL.

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Lab products found in correlation

Histrap crude column, by GE Healthcare (2 mentions) Profinia protein purification system, by Bio-Rad (1 mentions) 0.45 μm filter, by Merck Group (1 mentions) Ni nta beads, by Smart-Lifesciences (1 mentions) Mono q 5 50 gl column, by GE Healthcare (1 mentions) Dextrin beads 6ff, by Smart-Lifesciences (1 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions) Centrifugal filter, by Merck Group (1 mentions) Prep cell apparatus, by Bio-Rad (1 mentions) Protease inhibitor tablet, by Roche (1 mentions) Niso4, by GE Healthcare (1 mentions)

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Superdex 200 column (565 citations) Superdex 200 16/60 column (99 citations) HiLoad 16/60 Superdex 200 (90 citations) HiLoad 16/60 Superdex 200 column (85 citations) Superdex 200 size exclusion column (71 citations) Superdex 200 16/60 (44 citations) HiLoad Superdex 200 (24 citations) Superdex 200 16/60 size-exclusion column (8 citations) HiLoad Superdex 200 size exclusion column (5 citations) HiLoad Superdex size exclusion column (2 citations)

8 protocols using hiload 16 60 superdex 200 size exclusion column

Purification of E. coli Hfq Variants

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E. coli C-terminal His₆-tagged Hfq protein was expressed from pET15b vector (Novagen) and purified as previously described (Małecka et al. 2015 (link)). Plasmids for expression of Hfq R16A, Hfq Y25D, and Hfq K56A mutants were generated from the pET15b-hfq vector using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) with specific primers. After overexpression, cells were lysed by sonication. Then the lysate was applied to a HisTrap crude column (GE Healthcare) charged with NiSO₄ and the sample was eluted with imidazole gradient. The preparation was treated with RNase A (30 μg/mL) and DNase I (5 U/mL) for 1 h at 37°C to remove contaminating nucleic acids. Then it was purified again on a Ni²⁺ affinity column. Eluted fractions were concentrated and loaded onto HiLoad 16/60 Superdex 200 size exclusion column (GE Healthcare). The sample was eluted in the storage buffer (50 mM HEPES pH 7.5, 250 mM NH₄Cl, 1 mM EDTA, and 10% glycerol), and the Hfq concentration was determined from absorption at 280 nm as previously described (Olejniczak 2011 (link)).

Kwiatkowska J., Wroblewska Z., Johnson K.A, & Olejniczak M. (2018). The binding of Class II sRNA MgrR to two different sites on matchmaker protein Hfq enables efficient competition for Hfq and annealing to regulated mRNAs. RNA, 24(12), 1761-1784.

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Purification of Hfq Protein with RNase/DNase

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Hfq protein was expressed at 18°C for 16 hours. The lysis buffer and elution buffer contained 1 M NaCl instead of 150 mM and 500 mM NaCl, respectively. To remove the contaminating nucleic acids, the His tag affinity purified protein was treated with 30 μg/mL RNase A and 5 U/mL DNase I for 1 hour at 37° C, concentrated, and loaded onto a HiLoad 16/60 Superdex 200 size exclusion column (GE healthcare). The absence of RNase A was further validated by incubating RNA with the protein.

Nandana V., Rathnayaka-Mudiyanselage I.W., Muthunayak N.S., Hatami A., Mousseau C.B., Ortiz-Rodríguez L.A., Vaishnav J., Collins M., Gega A., Mallikaarachchi K.S., Yassine H., Ghosh A., Biteen J.S., Zhu Y., Champion M.M., Childers W.S, & Schrader J.M. (2023). The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions. bioRxiv.

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Recombinant Laforin Protein Purification

Cited 1 time

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H. sapiens (Hs) Laforin residues 1–328 was expressed from pET28b (Novagen) as an N-terminal His₆ tagged protein, as previously described^{31 (link)}. Briefly, laforin was expressed in BL21 (DE3) (Novagen) E. coli cells grown in 2xYT media at 37° C until OD₆₀₀ = 0.6, culture flasks were placed on ice for 20 min, induced with 1 mM (final) isopropyl thio-ß-D-galactopyranoside (IPTG), grown for an additional 14h at 20° C, and harvested by centrifugation. Cells were resuspended and lysed in buffer A (20 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5), centrifuged, and the proteins were purified using a Profinia immobilized metal affinity chromatography (IMAC) column with Ni²⁺ beads (Bio-Rad) and a Profinia protein purification system (Bio-Rad) using wash (buffer A) and elution buffer (300mM imidazole, 20 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5). The desalted elution fraction was further purified using fast protein liquid chromatography (FPLC) with a HiLoad 16/60 Superdex 200 size exclusion column (GE Healthcare). The buffer used for laforin purification for the small-scale pulldowns described in methods 2.3.2 was (50 mM HEPES, 100 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5. For all other experiments, laforin was purified in 20 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5.

Simmons Z.R., Sharma S., Wayne J., Li S., Vander Kooi C.W, & Gentry M.S. (2021). Generation and characterization of a laforin nanobody inhibitor. Clinical biochemistry, 93, 80-89.

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Purification of Hfq Protein with Affinity and Size Exclusion Chromatography

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Untagged Hfq was over-expressed as described (Santiago-Frangos et al., 2016 (link)) and purified with the following modifications: the cells were collected by centrifugation at 5,000 × g for 10 min, resuspended in 50 mL lysis buffer (50 mM Tris-HCl, pH 8, 500 mM NH₄Cl, 250 mM MgCl₂, 1 mM 2-mercaptoethanol) and lysed by sonication. The lysate was clarified by centrifugation (27,000 × g for 20 min at 4 °C), heated to 85°C for 45 min followed by another centrifugation step. The supernatant was treated with RNase A (30 μg/mL) and DNase I (5 U/mL) for 1 h at RT with shaking, following by filtration through a 0.45 μm filter (Millipore). The clarified lysate was applied to a 5 mL Hi-Trap column (GE Healthcare) charged with NiSO₄. The column was washed with 50 mM Tris-HCl pH 8, 500 mM NH₄Cl and 0.5 mM 2-mercaptoethanol, and then eluted with a linear gradient of the same buffer plus 0–1 M imidazole. Fractions containing Hfq were pooled and applied to a HiLoad 16/60 Superdex 200 size exclusion column (GE Healthcare) equilibrated with HB buffer (50 mM Tris-HCl 7.5, 250 mM NH₄Cl, 1 mM EDTA, 10% glycerol). The protein was concentrated with Amicon Ultra Centrifugal Filters (NMWL, 3 kDa) if needed, and stored in aliquots at −80 °C.

Małecka E.M, & Woodson S.A. (2021). Stepwise sRNA targeting of structured bacterial mRNAs leads to abortive annealing. Molecular cell, 81(9), 1988-1999.e4.

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Purification of Bacterial Fusion Proteins

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To express ZapE and K72A, the expression vector pET28a was used. The zapE and zapE_K72A each were amplified from PAO1 genomic DNA and pUCP20‐K72A with 28a‐zapE‐F and 28a‐zapE‐R. For PqsH, the expression vector pMAL‐C5x was chosen, and the pqsH was amplified with MAL‐pqsH‐F and MAL‐pqsH‐R. Gibson Assembly Master Mix (NEB) was applied to fuse the amplicons with the expression vectors. The fusion gene constructs were then transformed into E. coli strain BL21 (DE3). HIS‐ZapE and HIS‐K72A were purified on Ni NTA beads (Smart Lifesciences) independently
¹⁹ (link), and MBP‐PqsH was purified on Dextrin Beads 6FF (Smart Lifesciences) as previously reported
¹³ (link). The fusion proteins were further purified by ion exchange on a Mono Q 5/50 GL column (GE Biosciences) and then followed by a gel filtration on a HiLoad 16/60 Superdex 200 size exclusion column (GE Biosciences).

Liu X., Jia M., Wang J., Cheng H., Cai Z., Yu Z., Liu Y., Ma L.Z., Zhang L., Zhang Y, & Yang L. (2023). Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system. mLife, 2(1), 28-42.

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Purification of Tetrameric RII Protein

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Positive clones were electroporated into the E. coli BL21DE3 (star) expression cell line. A 1-L culture was grown in the presence of 100 μg/ml kanamycin at 37°C with shaking until the A₆₀₀=0.6. The culture was then switched to 23°C until the A₆₀₀=0.9, whereupon protein production was induced by the addition of 1 mM IPTG (isopropyl β-D-thiogalactoside) and growth was continued overnight at 23°C with shaking. The cell pellet was recovered by centrifugation and lysed by sonication in buffer containing 50 mM Tris–HCl (pH 9), 500 mM NaCl, and 2 mM CaCl₂. Cellular debris and insoluble matter were removed by centrifugation for 0.5 h at 16000 rpm in a JA25.5 rotor. The N-terminally 6× His-tagged protein was selected from other proteins by Ni-NTA affinity chromatography. The RII tetra-tandemer was then buffer-exchanged into a solution of 50 mM Tris–HCl (pH 9), 200 mM NaCl and 10 mM CaCl₂ using a centrifugal filter (Millipore). Concentrated protein was loaded onto a HiLoad 16/60 Superdex-200 size-exclusion column (GE Healthcare) for further purification. Fractions containing the tetra-tandemer were pooled and stored at 4°C for future use. Protein concentration was measured with a Nanodrop spectrophotometer (Thermal Fisher Scientific) and the purity was assessed by SDS/10%PAGE.

Vance T.D., Olijve L.L., Campbell R.L., Voets I.K., Davies P.L, & Guo S. (2014). Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice. Bioscience Reports, 34(4), e00121.

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Reconstitution of Histone Complexes

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The H2A–H2B and H3.1–H4 complexes were reconstituted as follows^{65 (link)}. Lyophilized H2A and H2B were mixed at a 1:1 molar ratio and dissolved in denaturing buffer (20 mM Tris-HCl (pH 7.5), 20 mM 2-mercaptoethanol, and 7 M guanidine hydrochloride). Lyophilized H3.1 and H4 were also mixed at a 1:1 molar ratio and dissolved in the same buffer. The mixtures were dialyzed against refolding buffer (10 mM Tris-HCl (pH 7.5), 5 mM 2-mercaptoethanol, 1 mM EDTA, and 2 M NaCl), and the H2A–H2B and H3.1–H4 complexes were refolded. The resulting complexes were purified by chromatography on a HiLoad16/60 Superdex 200 size-exclusion column (GE Healthcare), equilibrated with refolding buffer. For nucleosome reconstitution, the histone complexes (H2A–H2B and H3.1–H4) were mixed with a purified DNA fragment (the palindromic 146 base-pair alpha-satellite DNA or the 147 base-pair MMTV-A DNA). Nucleosomes were reconstituted by the salt-dialysis method, and were separately purified by native polyacrylamide gel electrophoresis using a Prep Cell apparatus (Bio-Rad)^{31 (link)}, under conditions with 20 mM Tris-HCl (pH 7.5) and 1 mM dithiothreitol.

Fujita R., Yamamoto T., Arimura Y., Fujiwara S., Tachiwana H., Ichikawa Y., Sakata Y., Yang L., Maruyama R., Hamada M., Nakao M., Saitoh N, & Kurumizaka H. (2020). Nucleosome destabilization by nuclear non-coding RNAs. Communications Biology, 3, 60.

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Purification of C-terminally His6-tagged E. coli Hfq

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C-terminally His6-tagged E. coli wild type (wt) Hfq protein and its mutants were expressed from pET15b vector (Novagen) and purified as described [38] . Cells were grown to OD600~0.5, induced with 1 mM IPTG (final concentration) followed by 3-4 h at 37ºC, and collected by centrifugation. Resulting pellet was resuspended in buffer A (50 mM HEPES, pH 7.5, 500 mM NH4Cl, 5% (w/v) glycerol) with protease inhibitor tablet (EDTA-free, Roche), and lysed by sonication. Resulting lysate was clarified by centrifugation and loaded onto HisTrap crude column charged with NiSO4 (GE Healthcare). Unbound proteins were washed out with buffer A supplemented with 10 mM imidazole. Proteins bound to the resin were eluted with a linear gradient of imidazole (35-600 mM) in the buffer A. To remove any residual nucleic acids, fractions containing Hfq protein were combined, concentrated, and treated with RNase A (30 µg/mL) and DNase I (5 U/mL) for 1 hour at 37°C. The solution was diluted to decrease the imidazole concentration and passed through a Ni 2+ affinity column. Fractions after elution were concentrated and applied to a HiLoad 16/60 Superdex 200 size exclusion column (GE Healthcare) equilibrated with the storage buffer (50 mM HEPES 7.5, 250 mM NH4Cl, 1 mM EDTA, and 10% glycerol). After elution, the protein was concentrated and stored in aliquots at -80ºC.

Małecka E.M., Sobańska D., & Olejniczak M. (2021). Bacterial chaperone protein Hfq facilitates the annealing of sponge RNAs to small regulatory RNAs.

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