Human serum albumin (recombinant expressed in rice) was purchased from Sigma-Aldrich, Saint Louis, MO (Sigma cat#A9731) and reconstituted in PBS. To covalently couple a fluorescent label to the reactive lysines of HSA, NHS-fluorescein (Thermo Fisher, Rockford, IL, cat# 46410), dissolved in DMSO, was mixed in 10-fold excess with the HSA at a pH of 7.4. This reaction took place at room temperature for 2 h in the dark. To quickly assess the efficiency of the reaction, the labeled protein was passed through a dye removal column (Thermo Fisher cat# 22858) according to manufacturer’s recommendations and the effluent was analyzed by absorbance at 450 and 280 nm. For microscopy and cell based assays, all labeled HSA preparations were purified using a AKTAprime Plus FPLC System (GE 11001313) using a HiLoad Superdex 200 PG (GE 28989335) column with PBS pH 7.4 as the buffer. One milliliter fractions were collected and screened using A280, A450, and SDS PAGE for the presence of relevant amounts of labeled HSA. Positive fractions were pooled and sterile filtered for downstream purposes.
Hiload superdex 200 pg
Manufactured by GE Healthcare
Sourced in United Kingdom
The HiLoad Superdex 200 pg is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It features a porous agarose-based matrix that allows for efficient separation based on molecular size. The column is compatible with a variety of buffers and can be used in both analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Dye removal column, by Thermo Fisher Scientific (1 mentions)
Nhs fluorescein, by Thermo Fisher Scientific (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Akta prime plus fplc system, by GE Healthcare (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
Kta prime chromatography system, by GE Healthcare (1 mentions)
Toyopearl phenyl 650 m, by Tosoh (1 mentions)
Akta pure fplc system, by GE Healthcare (1 mentions)
7 protocols using hiload superdex 200 pg
1
Fluorescent Labeling of Recombinant HSA
2
Histone Refolding and Purification
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Histones were refolded and purified as previously described with minor changes (9 (link), 33 (link)). Briefly, histone proteins were unfolded in 50 mM tris (pH 7.5), 100 mM NaCl, 10 mM dithiothreitol (DTT), and 6 M guanidine hydrochloride, mixed in equimolar ratios to a final protein concentration of 1 mg/ml, and then dialyzed at 4°C overnight to 10 mM tris-HCl (pH 7.5), 1 mM EDTA, 5 mM BME, and 2 M NaCl, followed by SEC at 4°C on a HiLoad Superdex 200 pg (GE Healthcare Life Sciences) column pre-equilibrated in the same buffer. For experiments using the stoichiometric core-histone mix (octamer-mix), the purified histone complexes were exchanged to assay buffer [25 mM NaPi (pH 7.0) and 300 mM NaCl] using a 10-kDa MWCO Amicon Ultra Centrifugal Filter Unit (Merck Millipore). Complexes were aliquoted, flash-frozen in liquid nitrogen, and stored at −20°C. Concentrations of H3-H4 are always given as concentration of dimers. Unless noted otherwise, concentrations of the octamer-mix are expressed as the equivalent histone octamer concentration, i.e., 10 μM octamer-mix equals 20 μM H2A-H2B and 20 μM H3-H4, corresponding to an equivalent histone octamer concentration of 10 μM.
3
Engineered Ferritin Mutants Expression
4
Purification of Recombinant Protein by FPLC
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The first step of protein purification by anion-exchange chromatography was performed as described previously (Ishibashi et al., 2005 ▶ , 2011 ▶ ). The soluble fraction of disrupted cells was applied onto a HiTrap Q HP column (1.6 × 2.5 cm, GE Healthcare) using an ÄKTAprime chromatography system (GE Healthcare). The bound proteins were eluted with a 100 ml linear gradient of NaCl from 0.3 to 0.9 M in 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 buffer. The fractions containing HaAP were pooled and dialyzed against 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 containing 3 M NaCl buffer. After dialysis, 30% ammonium sulfate was added and the soluble fraction was collected by centrifugation at 12 000g for 15 min. Proteins in the soluble fraction were loaded onto a hydrophobic column (30 ml, Toyopearl Phenyl-650M, Tosoh Bioscience) equilibrated with 50 mM Tris–HCl pH 8.0 containing 2 mM MgCl2, 0.5 M NaCl, 30% ammonium sulfate. The flowthrough fraction was then applied onto a gel-filtration column (1.6 × 60 cm, HiLoad Superdex 200 pg, GE Healthcare) equilibrated with 50 mM Tris–HCl pH 8.0, 2 mM MgCl2 buffer containing 0.5 M NaCl. The elution was pumped at 0.5 ml min−1. The protein purity was checked by 10% SDS–PAGE.
5
Purification of PnpM-His6 Protein
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The sample containing PnpM-His6 was loaded onto a column (1.6 × 60 cm) of Hiload Superdex 200 pg (FPLC system, GE Healthcare, Little Chalfont, UK) that was equilibrated with 10 mM of Tris-HCl buffer (pH 7.5, with 0.1 M NaCl and 5% glycerol) for 2 h at a flow rate of 0.5 ml min−1. The target protein was then eluted with the same buffer. The native molecular mass of PnpM was estimated from a calibration curve plotted by using the standard proteins (Zhang et al., 2009 (link)).
6
Synthesis of BAP Copolymers for ELISA Probes
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The SEC samples of BAP copolymers were prepared
according to the
same procedures for TL-catalyzed polymerization of proteins, which
are mentioned above. The molar ratio of BAPs to pG2pA-Y
was set at 100:1 (50–0.5 μM) with a total volume of 1
mL in 10 mM Tris-HCl, (pH 8.0). The concentration of TL was adjusted
to 5 μM to facilitate high copolymerization efficiency between
the BAPs and pG2pA-Y in a large volume. The polymerization
reaction was conducted at 37 °C for 18 h. The copolymer solution
was then injected into the SEC column (HiLoad Superdex 200 pg, GE
Healthcare). The SEC mobile phase was 10 mM Tris-HCl (pH 8.0). The
fractions containing polymers were pooled and concentrated using an
ultrafiltration membrane (30 kDa MWCO) and analyzed using SDS-PAGE
and native-PAGE. The enzymatic activity of the polymers was measured,
and they were functionally evaluated as probes in ELISA.
according to the
same procedures for TL-catalyzed polymerization of proteins, which
are mentioned above. The molar ratio of BAPs to pG2pA-Y
was set at 100:1 (50–0.5 μM) with a total volume of 1
mL in 10 mM Tris-HCl, (pH 8.0). The concentration of TL was adjusted
to 5 μM to facilitate high copolymerization efficiency between
the BAPs and pG2pA-Y in a large volume. The polymerization
reaction was conducted at 37 °C for 18 h. The copolymer solution
was then injected into the SEC column (HiLoad Superdex 200 pg, GE
Healthcare). The SEC mobile phase was 10 mM Tris-HCl (pH 8.0). The
fractions containing polymers were pooled and concentrated using an
ultrafiltration membrane (30 kDa MWCO) and analyzed using SDS-PAGE
and native-PAGE. The enzymatic activity of the polymers was measured,
and they were functionally evaluated as probes in ELISA.
7
Protein Purification for Crystallization
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The purity and size of the proteins were checked using SDS-PAGE and their concentrations determined using absorbance at 280 nm (NanoDrop 2000c; Thermo Scientific) and their molar extinction coefficients 52 .
Purification of proteins for crystallisation. IMAC-purified proteins were further purified by SEC using a HiLoad™ Superdex 200 pg on an AKTA Pure FPLC system (GE Healthcare Life Sciences). The purity of the fractions were determined using SDS-PAGE and those of high enough purity were pooled and concentrated to ~ 10 mg/ml.
Purification of proteins for crystallisation. IMAC-purified proteins were further purified by SEC using a HiLoad™ Superdex 200 pg on an AKTA Pure FPLC system (GE Healthcare Life Sciences). The purity of the fractions were determined using SDS-PAGE and those of high enough purity were pooled and concentrated to ~ 10 mg/ml.
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