6xHis-Rad6, 6xHis-Ubc4, Ubi-ProtA-6xHis, and UbiK48R-ProtA-6xHis were expressed in E.coli BL21(DE3) pRIL and purified over a pre-packed HisTrap FastFlow column (GE Healthcare). FLAG-Ufd4, FLAG-Ubr1, FLAG-Ufd2, and FLAG-Tom1 were overexpressed in yeast from a GPD promoter and purified as described (Hwang & Varshavsky, 2008 (link); Hwang et al, 2009 (link)). Purified yeast Uba1 and ubiquitin were purchased from BostonBiochem (#E-300 and #U-100SC, respectively). Final protein concentrations were 100 nM (Uba1), 80 μM (ubiquitin), 1 μM (Rad6), 1 μM (Ubc4), 200 nM (Ubr1), 200 nM (Ufd4), 200 nM (Ufd2), 200 nM (Tom1), 125 nM (Ubi-ProtA), 125 nM (UbiK48R-ProtA), in 20 μl reactions containing 4 mM ATP (#1191, Merck), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 50 mM Hepes (pH 7.5). All reactions contained Uba1, ubiquitin, Rad6, and Ubc4. Reactions were pipetted on ice, incubated at 30°C for 30 min, quenched by addition of 20 μl 5× SDS sample buffer (50% [vol/vol] glycerol, 10% [wt/vol] SDS, 250 mM Tris–HCl, pH 6.8, 62.5 mM EDTA, and 5% [vol/vol] β-mercaptoethanol) and incubation at 95°C for 5 min, and analyzed using 4–12% NuPAGE Bis-Tris gradient gels (Invitrogen) followed by immunoblotting with rabbit peroxidase–anti-peroxidase (PAP) antibodies (Z0113; Dako) and imaging on a LAS-4000 system (Fuji).
Histrap fast flow column
Manufactured by GE Healthcare
Sourced in China, United Kingdom
The HisTrap Fast Flow column is a chromatography column designed for the purification of histidine-tagged proteins. It is filled with a high-flow agarose resin that allows for efficient and rapid protein purification. The column utilizes the affinity of the histidine tag to the immobilized metal ions on the resin, enabling the selective capture and elution of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Serva Electrophoresis (2 mentions)
Sfm 900 media, by Thermo Fisher Scientific (2 mentions)
Sfm 900 methionine cysteine media, by Thermo Fisher Scientific (2 mentions)
Superdex 200 column, by GoldBio (2 mentions)
Nickel nta agarose beads, by GoldBio (2 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Nupage bis tris gradient gel, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Ubiquitin, by Merck Group (1 mentions)
Las 4000 system, by Fujifilm (1 mentions)
Las 4000, by Cytiva (1 mentions)
Finasteride, by Merck Group (1 mentions)
Hitrap blue hp column, by GE Healthcare (1 mentions)
Cortisone, by Steraloids (1 mentions)
Hitrap, by Cytiva (1 mentions)
Plasmid kits, by Qiagen (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Gf prefilter, by Sartorius (1 mentions)
Millex hp filter unit, by Merck Group (1 mentions)
13 protocols using histrap fast flow column
1
Reconstitution of Ubiquitin Conjugation Pathway
2
Purification and Immobilization of Fluorescent Proteins
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Proteins rsEGFP2 and DronpaM159T were expressed in Escherichia coli BL21-RILP from pQE31 expression vectors and purified via Ni-nitriloacetic acid (Ni-NTA) affinity chromatography (HisTrap Fast Flow column, GE Healthcare) following the manufacturer’s instructions. Purified proteins were washed and concentrated in 100 mM Tris-HCl and 150 mM NaCl pH 7.5 by ultrafiltration.
Ni-NTA was used to functionalize silica beads (Micromod Partikeltechnologie; 10 g l–1) which were incubated for 30 min with rsEGFP2 protein in 100 mM Tris-HCl and 150 mM NaCl pH 7.5. Protein concentration varied depending on the size of the beads, and was tuned to be proportional to their total surface area. In particular, starting from a stock of protein of 50 μM, the concentration was reduced to have one protein every 150 nm2 of total bead surface.
Samples were measured in 4-μl drops deposited on coverslips pretreated with bovine serum albumin 1% for 15 min, and washed twice with buffer. To avoid drying of the drops, a closed chamber with silicon barriers was used (Ibidi) with the addition of 1 ml of buffer inside one of the sectors.
Ni-NTA was used to functionalize silica beads (Micromod Partikeltechnologie; 10 g l–1) which were incubated for 30 min with rsEGFP2 protein in 100 mM Tris-HCl and 150 mM NaCl pH 7.5. Protein concentration varied depending on the size of the beads, and was tuned to be proportional to their total surface area. In particular, starting from a stock of protein of 50 μM, the concentration was reduced to have one protein every 150 nm2 of total bead surface.
Samples were measured in 4-μl drops deposited on coverslips pretreated with bovine serum albumin 1% for 15 min, and washed twice with buffer. To avoid drying of the drops, a closed chamber with silicon barriers was used (Ibidi) with the addition of 1 ml of buffer inside one of the sectors.
3
Purification of Spindly Protein Complex
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Expression of Spindly constructs was performed with the biGBac system 63 (link) in TnaO38 cells. Specically, the coding sequence of Spindly constructs were subcloned into the multiple cloning site of pLIB with a 6-histidine tag at the 5’end. Lysis of a pellet from 500 ml expression culture was performed by sonication in 100 ml lysis buffer (50 mM Hepes, pH 8.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole, 2mM TCEP, 1 mM PMSF, and 1 mM protease inhibitor cocktail [Serva]). The cleared lysate was loaded onto an equilibrated 5-ml HisTrap Fast Flow column (GE Healthcare) using a peristaltic pump (2 ml/min ow rate). The column was washed with 500 ml wash buffer (50 mM Hepes, pH 8.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole, and 2mM TCEP). Elution was performed with wash buffer supplemented with 250 mM imidazole. The 2-ml fractions were analyzed by SDS- PAGE, and those containing Spindly were concentrated up to a volume of 10 ml. The protein solution was diluted five times with buffer A and subsequently purified on a Resource Q anion exchange column. Peak fractions were analyzed by SDS-PAGE, and those containing Spindly constructs were concentrated up to 500 μl and applied to a Superose 6 10/300 column. The peak fractions were analyzed by SDS-PAGE, and those containing pure Spindly proteins were concentrated up to 20 mg/ml, flash frozen in liquid nitrogen, and stored at −80°C.
4
Purification of Baculovirus-Expressed PLD1
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PLD1 CatWT, PLD1 CatΔIns, and PLD1 CatΔIns mutants were expressed in Sf9 cells using baculovirus. 800 mL of cells were infected with 4 mL of baculovirus at 2–4 million cells/mL at >95% viability and harvested 48 hours later. Cell pellets were lysed in 50 mM Tris, pH 7.5, 400 mM NaCl, 5 mM Imidazole, 5% glycerol and the lysates centrifuged at 22,000 rpm and passed through a 0.22 μm filter. PLD1 protein was purified with a 5 mL HisTrap FastFlow column (GE Healthcare) followed by size exclusion chromatography using a HiLoad 26/600 Superdex 200 pg column in 20 mM Tris, pH 7.0, 150 mM NaCl. PLD1 ΔINS and WT PLD1 were concentrated to 2.79 and 0.91 mg/mL, respectively, flash-frozen, and stored at −80°C.
Selenomethionine-enriched PLD1 CatΔIns protein was expressed following the protocol described by Cronin et al.38 (link), using SFM-900 media (Invitrogen) with exchange to SFM-900 Methionine(−) Cysteine(−) media (Invitrogen) doped with selenomethionine 16 hours after baculoviral infection. Selenomethionine-enriched PLD1 CatΔIns protein was purified using the batch method. The filtered lysate was incubated with 2 mL nickel NTA agarose beads (Goldbio) for one hour and then applied to a column, washed with 30 mM imidazole buffer, eluted with 300 mM imidazole buffer, and finally applied to a Superdex 200 column.
Selenomethionine-enriched PLD1 CatΔIns protein was expressed following the protocol described by Cronin et al.38 (link), using SFM-900 media (Invitrogen) with exchange to SFM-900 Methionine(−) Cysteine(−) media (Invitrogen) doped with selenomethionine 16 hours after baculoviral infection. Selenomethionine-enriched PLD1 CatΔIns protein was purified using the batch method. The filtered lysate was incubated with 2 mL nickel NTA agarose beads (Goldbio) for one hour and then applied to a column, washed with 30 mM imidazole buffer, eluted with 300 mM imidazole buffer, and finally applied to a Superdex 200 column.
5
Cortisone and 7α-Hydroxycholest-4-en-3-one Synthesis
6
Purification of Spindly Protein Complex
7
Purification of Baculovirus-Expressed PLD1
8
Cloning and Expression of Proteins
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The expression vector pET-28a and pMo130-TelR plasmids were stored in our laboratory. A549 and THP1 cells were also from our laboratory. A HisTrap Fast Flow column (5 mL) was purchased from GE Healthcare (Beijing, China). Taq DNA polymerase and T4 DNA ligase were purchased from New England Biolab. PCR primers were synthesized by GENEWIZ (China). The bacteria genomic DNA extraction kits and plasmid kits were purchased from Qiagen. 55FeCl3 solution was from Perkin Elmer. Acinetobactin and preacinetobactin were synthesized by Sks Chem Company. All other chemicals and reagents were obtained from commercial sources and were of the highest purity available.
9
Recombinant Human Spindly Protein Expression
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Expression of human Spindly was performed with the biGBac system in TnaO38 cells. Lysis of a pellet from 500 ml expression culture was performed by sonication in 100 ml lysis buffer (50 mM Hepes, pH 8.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole, 5 mM β-mercaptoethanol [βMe], 1 mM PMSF, and 1 mM protease inhibitor cocktail [Serva]). The cleared lysate was loaded onto an equilibrated 5-ml HisTrap Fast Flow column (GE Healthcare) using a peristaltic pump (2 ml/min flow rate). The column was washed with 500 ml wash buffer (50 mM Hepes, pH 8.5, 200 mM NaCl, 10% glycerol, 20 mM imidazole, and 5 mM βMe). Elution was performed with wash buffer supplemented with 250 mM imidazole. The 2-ml fractions were analyzed by SDS-PAGE, and those containing Spindly were concentrated up to a volume of 10 ml. The protein solution was diluted five times with buffer A and subsequently purified on a Resource Q anion exchange column. Peak fractions were analyzed by SDS-PAGE, and those containing Spindly were concentrated up to 500 µl and applied to a Superose 6 10/300 column. The peak fractions were analyzed by SDS-PAGE, and those containing pure Spindly were concentrated up to 20 mg/ml, flash frozen in liquid nitrogen, and stored at −80°C.
10
Purification and Characterization of GP4-Tm and GFP:GP4-Tm Antigens
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The GP4-Tm and GFP:GP4-Tm antigens were purified by IMAC on an Äkta Explorer® purification system fitted with a His Trap® Fast Flow column (1ml, GE healthcare). Seed extracts were prepared in binding buffer (50 mM phosphate buffer pH 7.4, 0.3 M NaCl, 20 mM imidazole, 0.1% CHAPS supplemented with an EDTA-free proteinase inhibitor cocktail (1 tablet per 50 ml; Roche)) by adding frozen and pulverized seeds to cold buffer at a fixed w/v of 1 g seeds per 70 ml buffer. The extract was vortexed and centrifuged for 30 minutes at 4°C and 40,000 g to remove cell debris. The supernatant was removed, passed through a GF-prefilter (Sartorius) and a Millex®-HP filter unit (Millipore) and then flowed over the IMAC column. After washing the column with binding buffer, antigens were eluted with 0.5 M imidazole (in binding buffer). To remove the imidazole, the antigens were washed with PBS on Amicon® Ultra centrifugal filters (Millipore, 3 kDa molecular weight cutoff). Antigen purity was determined by densitometric analysis of Coomassie-stained polyacrylamide gels using the aforementioned imaging system and the Image Lab 3.0 software (Biorad). Antigen concentration was derived from the sample optical density at 280 nm measured by a NanoDrop® ND-1000 spectrophotometer.
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