NMR experiments were performed on a CatN /CatC complex (500 μM) enriched with 13C and 15N isotopes. The intein fragments were rendered inactive through CatN(C1A) and CatC(N134A) mutations. Polypeptides were individually expressed as SUMO fusion constructs in isotopically enriched M9 minimal media, purified and the fusion tag was removed by Ulp1 protease. Intein fragments were combined in 1.5:1 ratio of CatN: CatC and the complex formed was purified by size exclusion chromatography using a GE HiPrep 16/60 Sephacryl S-200 column. NMR experiments were performed on a Bruker DRX600 NMR spectrometer equipped with a TCI cryoprobe.
Hiprep 16 60 sephacryl s 200 column
Manufactured by GE Healthcare
The HiPrep 16/60 Sephacryl S-200 column is a size-exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. The column is packed with Sephacryl S-200 resin, which provides high resolution separation over a wide molecular weight range. The column dimensions are 16 mm in diameter and 60 cm in length.
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Lab products found in correlation
4 protocols using hiprep 16 60 sephacryl s 200 column
1
Structural Characterization of Cat/Cat Complex
2
Recombinant Mtr4p Protein Purification
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Recombinant Mtr4p was expressed in BL21 (DE3) competent E. coli cells (NEB) by auto-induction [35 (link)]. Cells were collected and frozen at -80°C. Cell pellets were resuspended and disrupted by sonication in equilibration buffer (50 mM sodium phosphate pH 7.4, 10 mM β-ME, 10% Glycerol, with cOmplete EDTA-free protease inhibitor cocktail (Roche)). Cell lysate was clarified by centrifugation at 40, 000 x g for 30 min at 4°C and loaded onto a column containing His60 Ni superflow Resin (Clontech). The resins were washed with 10-column volumes of equilibration buffer with 20 mM imidazole and eluted with 5-column volumes of the same buffer containing 300 mM imidazole. Purified protein subsequently underwent gel filtration chromatography on HiPrep 16/60 Sephacryl S-200 column (GE Healthcare) in gel filtration buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM DTT). Fractions with pure Mtr4p were collected and concentrated using Macrosep advance centrifugal devices (Pall Corporation). Protein aliquots were stored in a buffer containing 50 mM sodium phosphate pH 7.4, 10 mM β-ME and 20% Glycerol at -80°C.
3
Determining Oligomeric States of ToyJ and ToyJKL
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The oligomeric states of ToyJ and
ToyJKL were determined using
analytical size exclusion chromatography with a HiPrep 16/60 Sephacryl
S200 column (GE Healthcare) and gel filtration protein standard (BioRad
#151-1901) as indicated inFigures S1 and S2 . The enzymes and standards were injected into the column pre-equilibrated
with 0.02 M HEPES·NaOH (pH 7.4) and 0.15 M NaCl and eluted over
0.15 L at 0.1 mL/min. Elution volumes were determined using UV traces
and SDS-PAGE analysis.
ToyJKL were determined using
analytical size exclusion chromatography with a HiPrep 16/60 Sephacryl
S200 column (GE Healthcare) and gel filtration protein standard (BioRad
#151-1901) as indicated in
with 0.02 M HEPES·NaOH (pH 7.4) and 0.15 M NaCl and eluted over
0.15 L at 0.1 mL/min. Elution volumes were determined using UV traces
and SDS-PAGE analysis.
4
Purification of Crude Laccase Enzyme
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The crude laccase enzyme was precipitated from the culture media using ammonium sulfate precipitation technique. Briefly, the fungal culture was centrifuged at 8,500 × g for 15 min at 4°C and the resulting supernatant was collected. Solid (NH4)2SO4 up to 60% saturation was added slowly to the supernatant and incubated at 4°C overnight with constant stirring. The precipitated proteins were sedimented by centrifuging at 8,500 × g for 15 min at 4°C, and the obtained pellet was resuspended in 100 mL of 0.05 M Tris buffer at pH 7.5. The enzyme solution was then concentrated using a 30 kDa ultrafiltration membrane (Amicon Ultra-15; Merck Millipore, USA) and purified on an FPLC System (ÄKTA Start; GE Healthcare). The buffered protein solution was first run on an ion-exchange HiTrap Q FF column (GE Healthcare) equilibrated with 0.05 M NaCl in 0.05 M Tris buffer at pH 7.5 and eluted in a linear gradient of salt at a flow rate of 2.5 mL/min. Both the wash and elution fractions were analyzed for laccase activity, as described in the previous section. Fractions with the highest laccase activity were pooled, concentrated by ultrafiltration, loaded onto a gel filtration HiPrep 16/60 Sephacryl S-200 column (GE Healthcare), and eluted at a flow rate of 1.0 mL/min. The resulting fractions containing purified laccase were pooled, concentrated, and used for further analyses.
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