E. coli strain BL21 cells containing pGEX-6P-1-NCL were cultured at 37 °C in LB medium to an OD 600 of 0.8 then induced with 0.3 mM isopropyl β-D-thiogalactoside at 25 °C for 16 h. Cells were harvested and lysed by sonication. The GST-NCL proteins were purified by Glutathione Sepharose 4FF (GE healthcare).
Glutathione sepharose 4ff
Manufactured by GE Healthcare
Sourced in Germany, Sweden
Glutathione Sepharose 4FF is a pre-packed affinity chromatography medium designed for the purification of glutathione S-transferase (GST) fusion proteins. It consists of glutathione, a tripeptide, immobilized on Sepharose 4 Fast Flow. The medium provides a simple, efficient, and reversible method for the purification of GST-tagged recombinant proteins from crude cell lysates or other complex samples.
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Lab products found in correlation
Mono q 5 50 gl, by GE Healthcare (1 mentions)
Source 15 rpc, by Cytiva (1 mentions)
Ni nta magnetic agarose beads, by Qiagen (1 mentions)
Expressway cell free e coli expression system, by Thermo Fisher Scientific (1 mentions)
Streamline chelating resin, by GE Healthcare (1 mentions)
Streamline chelating, by GE Healthcare (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Talon metal affinity resin, by BD (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
10 protocols using glutathione sepharose 4ff
1
Recombinant GST-NCL Protein Purification
2
Recombinant Expression and Purification of α-Synuclein Protein
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The human full-length α-synuclein and Gln79-α-synuclein proteins were recombinantly expressed following procedures described recently [57 (link)]. Purification included Ni2+-chelating chromatography on a Streamline Chelating resin (Streamline Chelating, GE Healthcare Life Sciences, Uppsala, Sweden). Fractions containing the expression construct were subjected to a second purification step via a glutathione sepharose resin (Glutathione Sepharose 4FF, GE Healthcare Life Sciences). The removal of glutathione was achieved by overnight dialysis against buffer containing 100 mM NaCl, 30 mM Tris/HCl pH 7.6, 0.1 mM DTT and a membrane with 6–8 kDa cutoff. Separation of the GST- and His-tag from the α-synuclein sequence by a TEV protease cleavage left an native N-terminus [53 (link)] followed by cyclization of Gln79-α-synuclein to pGlu79-α-synuclein with QC overnight at room temperature. The fractions obtained were analyzed and subjected to reversed phase chromatography (Source 15 RPC, GE Healthcare Life Sciences), followed by lyophilization and anion exchange chromatography (MonoQ 5/50GL, GE Healthcare Life Sciences). The final buffer used for the experiments was 20 mM Tris/HCl, pH 7.0, containing 100 mM NaCl. The purity of the samples was assessed by SDS PAGE and mass spectrometry. Protein concentrations were determined using UV absorption at 280 nm.
3
Purification of His6-tagged WISP1, GST, and GST-Siah-1
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In vitro protein synthesis of His6-tagged WISP1, GST and GST-Siah-1 fusion protein was performed using Expressway cell-free E. coli expression system (Invitrogen, Waltham, CA, United States), followed by purification of His6-tagged WISP1 using Ni-NTA magnetic agarose beads (Qiagen, München, Germany) and of GST and GST-Siah-1 using glutathione-Sepharose 4FF (GE Healthcare, Piscataway, NJ, United States). The eluted proteins were analyzed by immunoblot as previously described.
4
Purification of Huntingtin Exon-1 Protein
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The expression vector pGEX-6P-1 containing exon-1 of HTT and encoding 52 glutamine residues (HTTQ52) was kindly provided by Zoya Ignatova, University of Hamburg, and used to express recombinant HTT for aggregation assays. HTTQ52 with N-terminal GST-tag followed by a polyhistidine tag was expressed in E. coli strain BL21 after induction with 400 μM IPTG at 24 °C for 4 h. After harvesting by centrifugation, cells were disrupted by enzymatic lysis and sonification. The first purification step was carried out through Ni2+-chelating chromatography on a Streamline Chelating resin (Streamline Chelating, GE Healthcare Life Sciences). Fractions containing the expression construct were further purified in a second step via a glutathione sepharose resin (Glutathione Sepharose 4FF, GE Healthcare Life Sciences). The removal of glutathione was achieved by dialysis overnight against buffer containing 50 mM Tris/HCl, 150 mM KCl, pH 8.5. Fractions of interest were lyophilized and stored at − 20 °C until usage. The purity of the samples was assessed by SDS-PAGE and mass spectrometry. Protein concentrations were determined using UV absorption at 280 nm.
5
MBP-NANOS2 Protein Purification and Interaction
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MBP-NANOS2 protein was expressed in the E.coli BL21 (DE3) strain and purified with Amylose Resin (New England Biolabs). GST-NEDD4 and GST proteins were expressed in the E.coli BL21 Star (DE3) strain. Bacterial pellets were sonicated in a binding buffer (25 mM HEPES-KOH [pH 7.4], 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 1 mM EDTA and 1 mM PMSF). The supernatants were mixed with 1 mg of MBP-NANOS2 for 2 h at 4 °C and then mixed with glutathione-sepharose 4FF (GE Healthcare) followed by further incubation for 2 h. The precipitates were separated by SDS–PAGE and analysed by western blotting with anti-NANOS2 antibody or by CBB (Coomassie brilliant blue) staining.
6
TXNIP Purification and Analysis
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For analysis by mass spectrometry in vivo, HEK 293 T cells were transfected with a plasmid expressing GST-fused TXNIP. After 24 h, the cells were harvested and lysed in buffer containing 0.5% Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM HEPES (pH 7.2) and complete protease inhibitor cocktail. After incubation for 30 min at 4 °C, the lysates were centrifuged at 16,000 × g for 20 min. The supernatants were incubated overnight at 4 °C with Glutathione Sepharose 4FF (GE Healthcare) and then centrifuged at 10,000 × g for 5 min. The supernatant was discarded and the resin was washed five times with lysis buffer. Bound proteins were eluted by boiling in LDS–PAGE loading buffer. For analysis by mass spectrometry in vitro, the purified TRX(C73A)–T–TXNIP(C36S/C49S/C120S/C170S/C205S/C267S) complex was incubated with 100 mM DTT for 1 h at room temperature and then injected into a Superdex 200 10/300 GL gel filtration column installed on an AKTA purifier FPLC system to obtain T–TXNIP(C36S/C49S/C120S/C170S/C205S/C267S) and TRX(C73A). The fractions containing T–TXNIP(C36S/C49S/C120S/C170S/C205S/C267S) were dialyzed against 50 mM Tris-HCl (pH 8.0), 500 mM NaCl and 10% glycerol to induce the formation of disulphide bonds between TXNIP molecules through air oxidation. Dialyzed proteins were then subjected to non-reducing SDS–PAGE.
7
Purification and Pull-down Assay of Ripply2
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His-tagged Ripply2 and the mutant proteins were expressed in E. coli, BL21, and purified with TALON Metal Affinity Resin (BD Bioscience, CA, USA). The GST pull-down method was described previously (Suzuki et al., 2012 (link)). Briefly, GST-tagged Tbx6 and the mutant proteins were expressed in the E. coli BL21 (DE3) strain and the bacterial pellets were sonicated in binding buffer (25 mM HEPES-KOH [pH 7.4], 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 1 mM EDTA, 1 mM PMSF), and then spun at 15,000 rpm at 4°C. The supernatants were mixed with His-Ripply2 and mixed with 30 ml of glutathione-sepharose 4FF (GE Healthcare, Sweden) followed by incubation for 2 hr. After extensive washing, precipitates were separated by SDS-PAGE and analyzed by western blotting with anti-Ripply2 antibody or by CBB staining.
8
GST Fusion Protein Expression and Purification
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For the construction of expression plasmids encoding glutathione-S-transferase (GST) fusion proteins, the cDNA fragment encoding the sea urchin ECM3 antigen
(a.a. 20-271) and sea urchin QBRICK antigen (a.a. 23-241) was amplified by PCR and cloned into pGEX 4T-1 (GE healthcare, Chicago, IL, USA). For bacterial
expression of GST-fusion proteins, the E. coli BL21 strain was transformed with individual expression plasmids and the expressed proteins were
affinity-purified using glutathione sepharose 4 FF (GE healthcare). Rats and guinea pigs were immunized with GST-fused QBRICK and ECM3 proteins, respectively.
Specific antibodies were purified by affinity purification using antigen-conjugated beads.
(a.a. 20-271) and sea urchin QBRICK antigen (a.a. 23-241) was amplified by PCR and cloned into pGEX 4T-1 (GE healthcare, Chicago, IL, USA). For bacterial
expression of GST-fusion proteins, the E. coli BL21 strain was transformed with individual expression plasmids and the expressed proteins were
affinity-purified using glutathione sepharose 4 FF (GE healthcare). Rats and guinea pigs were immunized with GST-fused QBRICK and ECM3 proteins, respectively.
Specific antibodies were purified by affinity purification using antigen-conjugated beads.
9
PANDA Protein Interaction Purification
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The PANDA CDS and the CDS of its interacting protein gene were cloned into the vectors pGSTA or pHISK for fusion with the GST or His tag, respectively. The GST or His fusion proteins were expressed in Escherichia coli BL21 and purified using Glutathione Sepharose 4 FF (#175132‐01 GE Healthcare, Chicago, Illinois) and NI‐NTA SefinoseTM Resin (C600033‐0010 BBI South Wales, UK) kits. Pull‐down assays were performed with the Pierce™ GST Protein Interaction Pull‐Down Kit (#21516 Thermo Scientifc, Waltham, Massachusetts).
10
Purification of Recombinant Human HSP90β
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A plasmid encoding human GST-HSP90β fusion protein (Zhang et al., 2014) was transformed into E. coli BL21 (DE3) codon plus cells. Cells were grown at 37°C in LB media until OD 600 of 0.6-0.8, then induced at 37°C with isopropyl β-d-1thiogalactopyranoside (IPTG, 1 mM) for 4 h. Cell pellets were collected by centrifugation and sonicated in PBS. Cell lysates were clarified by centrifugation at 17,700 x g. The supernatant was loaded onto Glutathione Sepharose 4 FF (GE healthcare) for affinity purification. Bound protein was cleaved by precision-protease at 4°C overnight, and HSP90β was collected in the flow-through fraction. Protein purity was confirmed following SDS-PAGE and Coomassie blue staining.
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