Chemicals, biochemicals, buffers, solvents, and the components for Luria-Bertani (LB) media were obtained from sources reported elsewhere.13 The syntheses of 2-hydroxy-2,4-pentadienoate (2 ) is reported elsewhere.9 (link) The 5-methyl derivative (6 ) was generated from 5-(methyl)-2-hydroxymuconate14 following the procedure used to produce 2 .9 (link) The Phenyl Sepharose 6 Fast Flow, DEAE-Sepharose resins, and the prepacked PD-10 Sephadex G-25 columns were obtained from GE Healthcare (Piscataway, NJ). The Econo-Column chromatography columns and Freeze ‘N Squeeze units were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Enzymes and reagents used for molecular biology procedures were obtained from New England Biolabs, Inc. (Ipswich, MA). 5-(carboxymethyl)-2-Hydroxymuconate isomerase (CHMI),15 4-oxalocrotonate tautomerase (4-OT)16 (link),17 (link), 4-OD/VPH and 4-OD/E106QVPH from P. putida mt-2 were purified by procedures reported elsewhere with some modifications.9 (link) The 4-OD/VPH complex from L. cholodnii SP-6 was purified similarly to the P. putida mt-2 complex.9 (link) Activities were determined using previously described assays.9 (link),15 –17 (link)
Phenyl sepharose 6 fast flow
Manufactured by GE Healthcare
Sourced in United States
Phenyl Sepharose 6 Fast Flow is a chromatography resin used for the purification of biomolecules. It is a hydrophobic interaction chromatography medium composed of highly cross-linked agarose beads substituted with phenyl groups. The resin is designed for fast flow separations and can be used for the purification of proteins, peptides, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Econo column chromatography column, by Bio-Rad (2 mentions)
Prepacked pd 10 sephadex g 25 columns, by GE Healthcare (1 mentions)
Ultrafiltration membrane, by Merck Group (1 mentions)
Propiolic acid, by Merck Group (1 mentions)
Sephadex g 75, by GE Healthcare (1 mentions)
Deae sephadex, by GE Healthcare (1 mentions)
Amicon stirred cell concentrators, by Merck Group (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
5 protocols using phenyl sepharose 6 fast flow
1
Enzymatic Synthesis and Purification
2
Purification and Characterization of N2 Enzyme
3
Purification of G. hollisae Protein
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The G. hollisae strain ATCC 33564 was obtained in a freeze-dried form from the Culture Collection and Research Center of the Food Industry Research and Development Institute (Hsin-Chu, Taiwan). Phenyl Sepharose 6 Fast Flow and protein molecular weight standards were purchased from GE Healthcare (Piscataway, NJ, USA). The protein assay kit was obtained from Bio-Rad (Hercules, CA, USA). Protein purification chemicals were obtained from Calbiochem (La Jolla, CA, USA).
4
Purification of Native Linker Histone H1
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Native linker histone was purified from 0–12 h after egg laying D. melanogaster embryos as previously decribed [77] (link) with the following modifications. The H1-containing supernatant of the second ammonium sulphate precipitation was subjected to phenyl sepharose chromatography (column volume: 20 ml; Phenyl Sepharose 6 Fast Flow; GE Healthcare). The pooled H1-containing fractions were subjected to extensive dialysis against 25 mM Hepes-KOH pH 7.6, 100 mM KCl, 0.1 mM EDTA, 10% glycerol, 5 mM β-mercaptoethanol. For the final cation exchange step, a Mono S column was used. H1-containing fractions were pooled, concentrated (Amicon Ultra-4 Centrifugal Filter Units 10 kDa; Millipore), and glycerol was added to 50% for storage at −20°C. H1 concentration was determined by Coomassie staining of SDS gels taking BSA as a reference. Band intensities were quantified using the Odyssey Infrared Imaging System (LI-COR). The yield from 60 g of embryos was ∼1 mg of H1.
5
Recombinant CaM7 and CML25 Purification
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Arabidopsis CML25 (AT1G24620) was amplified by PCR with the primer pair 5′-GAATTCCATATGATGTTCA ACAAAAACCAAGGATC-3′ and 5′-CCGCTCGAGCTA CCTCGGACCACCTCCCAT-3′, and CaM7 (AT3G43810) was amplified using primer pair 5′-GGAATTCCATATGA TGGCGGATCAGCTAACCGAT-3′ and 5′-CCGCTCGAG TCACTTTGCCATCATGACTTTGAC-3′. The sequenced PCR products were introduced into the Nde I and Xho I sites of the prokaryotic expression vector pET30a(+), and confirmed by DNA sequencing. The generated pET30a : CaM7 and pET30a : CML25 plasmids were transformed into E. coli strain BL21 (DE3). The transformants were grown at 37 °C until OD600 = 0.6 and subsequently induced with 0.4 mm isopropyl-b-D-thiogalactopyranoside for 10 h at 28 °C. Recombinant proteins were purified by Ca 2+ -dependent phenyl-sepharose chromatography (Amersham Bioscience, Phenyl Sepharose 6 Fast Flow, GE Healthcare) as described (Zielinski 2002) . The electrophoresis mobility shift assay for Ca 2+ binding was performed as described (Garrigos et al. 1991) using denatured protein incubated with either 5 mm CaCl2 or 5 mm EGTA (Ethylene Glycol Tetraacetic Acid).
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