SPR measurements were performed on a ProteOn XPR36 instrument (BioRad). Hexon protein was purified from HAdV5HVR7-infected HEK293 cells with AIEX using DEAE and MonoQ columns (GE Healthcare)69 (link) and biotinylated using NHS-biotin (Pierce). The biotinylated hexon was immobilized on a NeutrAvidin NLC sensor chip (BioRad) until 799 resonance units were reached. Purified scFcs (1 to 32 nM) were injected at a flow rate of 60 μL/min for 240 s. Dissociation was followed over at least 600 s. Simple Langmuir kinetic fitting was applied where appropriate (see Results) with the ProteOn Manager software, otherwise a heterogeneous ligand model was used.
Monoq columns
Manufactured by GE Healthcare
Sourced in United States, Sweden
MonoQ columns are ion exchange chromatography columns used for the purification and separation of biomolecules. They feature a strong anion exchange resin that can bind and separate various negatively charged molecules, such as proteins, nucleic acids, and other biomolecules, based on their charge differences.
Automatically generated - may contain errors
Lab products found in correlation
Neutravidin nlc sensor chip, by Bio-Rad (1 mentions)
Proteon xpr36, by Bio-Rad (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Mouse anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Desalting column, by GE Healthcare (1 mentions)
Phusion polymerase, by Thermo Fisher Scientific (1 mentions)
Dpni, by New England Biolabs (1 mentions)
3 protocols using monoq columns
1
Hexon Protein SPR Binding Assay
2
Purification and Analysis of Recombinant Proteins
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ATP and recombinant murine IL-2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). The tissue culture sera and media were purchased from Gibco (Carlsbad, CA, USA). Mouse anti–β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Q-Sepharose and monoQ columns were purchased from GE Healthcare (Waukesha, WI, USA). The proteasome inhibitor carbobenzoxy-l -leucyl-l -leucyl-l -leucinal (MG132) was purchased from Calbiochem (San Diego, CA, USA), and [(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino] propanoyl} amino)butyl] boronic acid (bortezomib, PS-341) was obtained from LC Laboratories (Woburn, MA, USA). The reagents for ECL were obtained from Bio-Rad (Hercules, CA, USA) and GE Healthcare. Bovine MBP was isolated as described (16 (link)). Most other chemicals were reagent grade and acquired from either Sigma-Aldrich or Helicon (Moscow, Russian Federation).
3
Protein Expression and Purification
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All chemicals were purchased from Merck KGaA (Darmstadt, Germany), Chemie Brunschwig AG (Basel, Switzerland), VWR (Hannover, Germany) or Carl Roth (Karlsruhe, Germany), and were used without further purification unless otherwise specified. NADPH was obtained from OYC Europe (Rotterdam, Netherlands) and also used without further purification. Phusion polymerase was purchased from Thermo Fisher Scientific (Waltham, MA, USA). DpnI was provided by New England Biolabs (Ipswich, MA, USA), and primers were ordered from Microsynth AG (Balgach, Switzerland). The Mono Q columns and the desalting columns were ordered from GE Healthcare (Uppsala, Sweden).
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