Hiprep 16 60 sephacryl s 100 hr column

Manufactured by GE Healthcare

Sourced in United States, Sweden

The HiPrep 16/60 Sephacryl S-100 HR column is a size-exclusion chromatography column used for the separation and purification of biomolecules. The column is packed with Sephacryl S-100 HR, a cross-linked copolymer of allyl dextran and N,N'-methylenebisacrylamide, which provides a porous matrix for the separation of proteins, peptides, and other macromolecules based on their size and molecular weight.

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Lab products found in correlation

Opti mem serum free medium, by Thermo Fisher Scientific (2 mentions) Amicon ultra 0.5 ml centrifugal filter, by Merck Group (2 mentions) Dnase, by Roche (2 mentions) Preparative chromatography system, by Waters Corporation (2 mentions) Preparative pump, by Waters Corporation (2 mentions) W717autosampler, by Waters Corporation (2 mentions) Kta fplc system, by GE Healthcare (1 mentions) Hitrap sp hp cation exchange column, by GE Healthcare (1 mentions) Bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Prsetb, by Thermo Fisher Scientific (1 mentions) Cgamp, by InvivoGen (1 mentions) Hilyte fluor 488, by AnaSpec (1 mentions) Hitrap q sepharose hp column, by GE Healthcare (1 mentions) Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (1 mentions) Hitrap heparin hp column, by GE Healthcare (1 mentions)

12 protocols using hiprep 16 60 sephacryl s 100 hr column

Optimized SEC Analysis of Aptamers

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Size-exclusion chromatography (SEC) experiments were performed using an ÄKTA FPLC system (GE Healthcare) with a HiPrep 16/60 Sephacryl S-100HR column (GE Healthcare). The column was equilibrated with 3 column volumes (CV) of running buffer (10 mM sodium phosphate, 100 mM NaCl, pH 7.4) prior to experiments. 100 μL of sample was injected onto the column and experiments were run at 0.5 mL min⁻¹ and continued for 2 CV. Elution was monitored by the eluate UV absorbance at 254 nm. Experiments were recorded and analyzed using the provided Unicorn 4.11 software package (GE Healthcare).
Aptamer samples were prepared by diluting the appropriate aptamer(s) in running buffer to a final concentration of 20 μM and immediately heated in a boiling water bath for 3 minutes and cooled in an ice bath to allow the aptamer to anneal. If quinine was to be added to the samples, quinine would be diluted in the sample to a final concentration of 0.1 mM prior to the annealing step.

Neves M.A., Slavkovic S., Reinstein O., Shoara A.A, & Johnson P.E. (2019). A proof of concept application of aptachain: ligand-induced self-assembly of a DNA aptamer. RSC Advances, 9(3), 1690-1695.

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Recombinant Mfsap1 Protein Production

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MFSAP1 gene was PCR amplified and cloned in Pichia pastoris GS115 following the condensed transformation method (68 (link)). Culture supernatants were collected after 72 h after methanol induction, and native Mfsap1 was enriched using Amicon 30 kDa ultra-centrifugal filter (Merck Millipore) and buffer exchanged into 20 mM sodium acetate buffer pH 4.2. Protein was subsequently purified through ion-exchange chromatography with HiTrap SP-HP cation exchange column (GE Healthcare) and size-exclusion chromatography with HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare). SEC fractions ranging from 25 and 45 kDa were collected and enriched using Amicon 10 kDa ultra-centrifugal filter (Merck Millipore) before protein was aliquoted and snap-freeze in liquid nitrogen and stored in −80°C.

Goh J.P., Ruchti F., Poh S.E., Koh W.L., Tan K.Y., Lim Y.T., Thng S.T., Sobota R.M., Hoon S.S., Liu C., O’Donoghue A.J., LeibundGut-Landmann S., Oon H.H., Li H, & Dawson TL J.r. (2022). The human pathobiont Malassezia furfur secreted protease Mfsap1 regulates cell dispersal and exacerbates skin inflammation. Proceedings of the National Academy of Sciences of the United States of America, 119(49), e2212533119.

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Characterization of IL-33 Isoforms

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Necrosis was induced by cryoshock in pools of TE-7 cells with Dox-inducible overexpression of WT or truncated IL-33 as described above. Size-exclusion chromatography was run on supernatants using a GE Healthcare HiPrep 16/60 Sephacryl S-100 HR column. Protein concentration was continuously monitored by measuring UV absorbance at a wavelength of 280 nm. Fractions of 1 mL were collected. IL-33 expression in fractions was determined by IL-33 ELISA (DY3625, R&D). In separate experiments, double-stranded DNA concentration in fractions was quantitated by Qubit (Q32854, Thermo Scientific) following the manufacturer’s instructions. Fractions corresponding to elution volumes of 35–39 and 55–59 mL were then pooled, concentrated by acetone precipitation, and subjected to western blot analysis.

Travers J., Rochman M., Miracle C.E., Habel J.E., Brusilovsky M., Caldwell J.M., Rymer J.K, & Rothenberg M.E. (2018). Chromatin regulates IL-33 release and extracellular cytokine activity. Nature Communications, 9, 3244.

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Chitosan-based Cytotoxicity Evaluation

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Low molecular weight chitosan (DD 85%) and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich Co (Saint-Louis, MO, USA). The bicinchoninic Acid (BCA)Protein Assay kit was purchased from Thermo Fischer Scientific (Waltham, MA, USA). A Hiprep 16/60 Sephacryl S-100 HR column was purchased from GE Healthcare Bio-science (Uppsala, Sweden), and a LUNA phenyl-hexyl column 250 × 4.6 × 5 μm was purchased from Phenomenex (Torrance, CA, USA). All other chemical reagents used in this study were purchased commercially and were of adequate analytical grade. B. toyonensis were provided by the Biochemistry Engineering Laboratory from the Federal University of Rio Grande do Norte (UFRN), under the following registration number at the National System of Genetic Resource Management and Associated Traditional Knowledge (SisGen): AD8AE98/Nov 2018.

da Silva N.S., Araújo N.K., Daniele-Silva A., Oliveira J.W., de Medeiros J.M., Araújo R.M., Ferreira L.D., Rocha H.A., Silva-Junior A.A., Silva M.S, & Fernandes-Pedrosa M.D. (2021). Antimicrobial Activity of Chitosan Oligosaccharides with Special Attention to Antiparasitic Potential. Marine Drugs, 19(2), 110.

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Purification and Thermal Shift Assay of STING

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Assays of the molecular interaction between the purified human STING C-terminal domain (amino acids 137 to 379; non-transmembrane domain) and M04 were performed as previously reported (19 (link)). Briefly, the 6xHis STING CTD open reading frame was cloned into pRSET-B (Invitrogen) and used to transform the Escherichia coli strain pLysS (Promega). The transformed E. coli cells were then induced to express the protein as induced by 1 mM IPTG (isopropyl-D-thiogalactopyranoside) at 16°C for 18 h. STING protein was purified by nickel-affinity chromatography (Clontech Laboratories) and then further purified by gel filtration chromatography (HiPrep 16/60 Sephacryl S-100 HR column; GE Healthcare Life Sciences). Eluted proteins were concentrated using Amicon centrifugal filters (10-kDa cutoff). For thermal shift assays, SYPRO Orange dye was used, following the manufacturer's suggested protocol, to determine protein stability in the presence and absence of cGAMP (Invivogen) or M04.

Abraham J., Botto S., Mizuno N., Pryke K., Gall B., Boehm D., Sali T.M., Jin H., Nilsen A., Gough M., Baird J., Chakhtoura M., Subra C., Trautmann L., Haddad E.K, & DeFilippis V.R. (2020). Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner. Frontiers in Immunology, 11, 1430.

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Isolation and Characterization of TE-7 Cell Secretome

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TE-7 cells were stimulated in OPTI-MEM serum-free medium (31985062; Gibco), and supernatant samples were collected. Samples were treated with DNAse (10104159001; Roche) and concentrated using 10-kDa Amicon Ultra-0.5 mL Centrifugal Filters (UFC501096; Millipore). Size exclusion chromatography was run on supernatants using a GE Healthcare HiPrep 16/60 Sephacryl S-100 HR column (GE17–1165-01; GE). The protein concentration was continuously monitored by measuring UV absorbance at a wavelength of 280 nm. Fractions of 1 mL were collected. Fractions were concentrated as described above, and the protein expression in eluates was determined by immunoblotting as described above.

Brusilovsky M., Rochman M., Rochman Y., Caldwell J.M., Mack L.E., Felton J.M., Habel J.E., Porollo A., Pasare C, & Rothenberg M.E. (2021). Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation. Nature immunology, 22(10), 1316-1326.

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Amyloid-beta and α-Synuclein Protein Labeling

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Human β-amyloid (1-42) (Aβ42), synthesized via solid-phase chemistry, was purchased from ERI Amyloid Laboratory (Oxford, CT, USA). Aβ42, conjugated with HiLyte Fluor 488 at the N-terminus, was purchased from AnaSpec (Fremont, CA, USA). Alexa Fluor 647 NHS ester, for N-terminal αS labeling, was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Aβ sequence-specific antibodies, 6E10, 4G8, and 12F4 were purchased from Biolegend (San Diego, CA, USA). The anti-Aβ(22-35) antibody was purchased from Sigma-Aldrich (St Louis, MO, USA). αS sequence-specific antibodies, F11, 5C2, 211 and D10, were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA) and Novus International (Saint Charles, MO, USA). Prepacked FPLC columns were purchased from GE Healthcare (Piscataway, NJ, USA), including HiTrap Q Sepharose HP column for anion exchange, HiPrep 16/60 Sephacryl S-100 HR column for size exclusion, and HiPrep 26/10 Sephadex column for desalting. All other chemical reagents were purchased from Fisher Scientific (Pittsburg, PA, USA) unless stated otherwise.

Chau E, & Kim J.R. (2022). α-synuclein-assisted oligomerization of β-amyloid (1-42). Archives of biochemistry and biophysics, 717, 109120.

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Isolation and Characterization of TE-7 Cell Secretome

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Casein Hydrolysate Peptide Fractionation

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In order to obtain peptide fractions from casein hydrolysates, gel filtration
throughout a preparative chromatography system performed (Waters, Milford, MA,
USA).
Peptides that identified in SDS-PAGE separated on a preparative scale for further
study. The preparative pump (Waters) and preparative liquid chromatography W600
(Waters) used with a Dual λ Absorbance detector W2487 (Waters) and W717
Autosampler (Waters).
The casein hydrolysates dissolved in 5 mM sodium phosphate buffer with 0.15 M
NaCl (Sigma), pH 7.0. It filtered with a PVDF 0.45 μm sterile syringe
membrane filter (Futecs). After that, casein hydrolysates is loaded 3.0 mL
throughout a Hiprep 16/60 Sephacryl S-100 HR column (GE Healthcare Life
Sciences, Marlborough, MA, USA) and eluted at flow rate of 1.0 mL/min. A
detector was set at 280 nm.
All fractions obtained throughout a gel filtration with Hiprep 16/60 Sephacryl
S-100 HR column on preparative chromatography system collected and fractions
stored at –20°C (Table
1).

Kim D.Y., Yoo J.S., Cho Y.A., Yoon H.S, & Kim C.H. (2021). Calcium Solubilization Ability and Anti-Inflammatory Effects of Hydrolyzed Casein. Food Science of Animal Resources, 41(4), 687-700.

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Purification of Insect-Derived Protein

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Culture supernatant from the wild-type P. entomophila was fractionated using ÄKTA start with a HiPrep 16/60 Sephacryl S-100 HR column (GE Healthcare). Each fraction was incubated with the wild-type recombinant. The sample from fraction No. 26 was dialyzed with 20 mM Tris-HCl, pH 7.5, and applied to a DEAE Sepharose CL-6B column (1×2 cm). The flow-through fraction was applied to a CM Sepharose CL-6B column (1×2 cm). After washing with 20 mM Tris-HCl, pH 7.5, the protein was eluted with a linear NaCl gradient (100–500 mM) in the same buffer.

Shibata T., Maki K., Hadano J., Fujikawa T., Kitazaki K., Koshiba T, & Kawabata S.I. (2015). Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins. PLoS Pathogens, 11(10), e1005244.

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