The following supplied the respective material requirements listed: New England BioLabs, USA, protein markers; Cambio, Cambridge, UK, PCMV-Sport plasmid with hGH; Sigma Aldrich, UK, phenyl-Sepharose, imidodiacetic-Sepharose, enterokinase, protein markers, monoclonal anti-hGH antibody secondary peroxidase-conjugated antibody and general analytical grade reagents; Fluka, Dansyl-D-Tyr-Val-Gly; monoclonal antibody to bGH and a monoclonal to cross-linked rabbit liver MTI/MT-II were obtained from Stratech, Newmarket, U.K., and tetrahydrobiopterin from Schircks Laboratories, Jona, Switzerland.
Phenyl sepharose
Manufactured by Merck Group
Sourced in United Kingdom, United States, Germany, Australia
Phenyl-Sepharose is a chromatographic resin composed of porous agarose beads with covalently attached phenyl groups. It is commonly used for the purification of proteins and other biomolecules based on hydrophobic interaction chromatography (HIC) principles. The phenyl groups on the resin surface provide a hydrophobic environment that can facilitate the selective binding and separation of target analytes from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Dna ladder, by New England Biolabs (1 mentions)
Dna gel extraction kit, by Qiagen (1 mentions)
Sephacryl s 200 hr, by Merck Group (1 mentions)
Ni nta agarose beads, by GE Healthcare (1 mentions)
Dntps mix, by New England Biolabs (1 mentions)
Oligonucleotide, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Q sepharose, by GE Healthcare (1 mentions)
Phenyl sepharose, by GE Healthcare (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
5 protocols using phenyl sepharose
1
Purification and Detection of hGH
2
Purification and Degradation of C. crescentus Proteins
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C. crescentus ClpX and ClpP were purified as previously described (Rood et al., 2012 (link)). ClpA* is a variant of C. crescentus ClpA lacking the final 9 residues that was shown with the E. coli ClpA orthologue to be fully active, but less prone to autodegradation (Maglica et al., 2008 (link)). Briefly, untagged ClpA* was purified via ammonium precipitation, followed by phenyl-sepharose (Sigma-Aldrich), anion-exchange (Q Fast Flow and Mono-Q; GE Healthcare), and size-exclusion (Sephacryl-200; GE Healthcare) chromatography. FtsZ was purified as previously described (Thanbichler and Shapiro, 2006 (link)). TAP–FtsA-MTS was purified using standard Ni-NTA purification (Thermo-Fisher). ClpXP and ClpAP degradation reactions were performed as previously described (Maglica et al., 2008 (link); Rood et al., 2012 (link)). Detailed purification and reaction protocols are available upon request.
3
Recombinant Protein Purification Protocol
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All chemicals used in this study were of analytical reagent grade. Ampicillin, isopropyl-β-D-1-thiogalactopyranoside (IPTG), phenyl sepharose, sephacryl S-200 HR, oligonucleotides and other chemicals were purchased from Sigma-Aldrich, Co. (St. Louis, USA). Restriction enzymes, DNA ladders, dNTPs mix and other material used for cloning were obtained from New England Biolabs Inc. (MA, USA). Ni-NTA agarose beads, SDS-PAGE molecular weight marker were purchased from GE Healthcare (UK). DNA gel extraction kit was procured from Qiagen Inc.(CA, USA).
4
Isolation and Culture of Human Adipose-Derived Stem Cells
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Human adipose derived stem cells (hADSC) were delivered by iXCells Biotechnologies (San Diego, USA, Cat. Nr. 10HU-001). RPMI 1640 medium was obtained from GIBCO (Thermo Fisher Scientific, Schwerte, Germany). Fetal bovine serum (FBS) was provided by HyClone Laboratories Inc. (Bath, UK). The sources of the phospholipases and antibodies (including the dilutions used) were given previously [53 (link)] unless indicated otherwise. 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS, premium grade) were bought from Pierce/Thermo Scientific (Rockford, IL, USA).
Protein A-Sepharose (Cl-4B) and phenylsepharose were from Calbiochem/Merck (Darmstadt, Germany). Polystyrene Bio-Beads SM-2 (20–50 mesh) were bought from Bio-Rad Laboratories (Munich, Germany). Mannosamine (ManN, 2-amino-2-deoxy D-mannose), methyl-ß-cyclodextrin (mßCD), BSA (fraction V, defatted) and human serum (normal healthy probands) were delivered by Sigma-Aldrich (Deisenhofen, Germany). Human (recombinant) insulin was a kind gift from Sanofi Pharma Germany GmbH (Diabetes group, Frankfurt am Main, Germany). Other materials (highest purity available) were obtained as described previously [31 (link),32 (link),53 (link),82 (link),83 (link)].
Protein A-Sepharose (Cl-4B) and phenylsepharose were from Calbiochem/Merck (Darmstadt, Germany). Polystyrene Bio-Beads SM-2 (20–50 mesh) were bought from Bio-Rad Laboratories (Munich, Germany). Mannosamine (ManN, 2-amino-2-deoxy D-mannose), methyl-ß-cyclodextrin (mßCD), BSA (fraction V, defatted) and human serum (normal healthy probands) were delivered by Sigma-Aldrich (Deisenhofen, Germany). Human (recombinant) insulin was a kind gift from Sanofi Pharma Germany GmbH (Diabetes group, Frankfurt am Main, Germany). Other materials (highest purity available) were obtained as described previously [31 (link),32 (link),53 (link),82 (link),83 (link)].
5
Recombinant sAPPα Production in Pichia
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Recombinant human sAPP695α was expressed in the methylotrophic yeast Pichia pastoris strain GS115 and purified from culture media by FPLC (BioRad) as previously described [26] (link). Chromatographic purification was by anion exchange using Q Sepharose (1.6×25 cm column, GE Healthcare) followed by hydrophobic exchange with phenyl Sepharose (1.6×25 cm column, GE Healthcare). APP eluted in phenyl Sepharose buffer B (50 mM Na2HPO4, pH 7) was then concentrated using Amicon Ultra-15 Centrifugal Filter Units (30 kDa, Merck Millipore, Australia) and stored at –80°C as 20 µM stocks. Elimination of anionic buffer in original sAPPα, APP, CP and BSA stocks was carried out by buffer-exchange with 50 mM HEPES, 150 mM NaCl, pH 7.2 (HBS) using a Superdex 200 10/GL filtration column (GE Healthcare).
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