From E. faecalis, acpA and acpB genes were cloned into pET-28a and pET-21a vectors, respectively (Novagen, USA) and the recombinant vectors were transformed into E. coli BL21 (DE3) cells. For NMR experiments, we labeled the proteins and 15N-labeled proteins were purified as described previously [13 (link)]. Using chelating sepharose HP (GE Healthcare, Sweden) with an imidazole gradient in 20 mM Tris buffer at pH 8.0. In case of EfAcpA, the N-terminal His-tag from EfAcpA was cleaved with 0.3% (w/w) thrombin at 25°C for 16 h and the protein was further purified using HiTrap QFF (GE Healthcare). In case of EfAcpB, protein was purified using HiTrap QFF and Resource Q columns (GE Healthcare).
Hitrap q ff
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom, United States
HiTrap Q FF is a pre-packed anion exchange chromatography column designed for rapid and efficient purification of biomolecules. It features a strong anion exchange matrix that can be used for the capture and separation of negatively charged proteins, peptides, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (28 mentions)
Superdex 75, by GE Healthcare (3 mentions)
Ni nta, by Qiagen (2 mentions)
Hitrap sp ff, by GE Healthcare (2 mentions)
α 32p gtp, by PerkinElmer (2 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Akta avant, by GE Healthcare (1 mentions)
Emulsiflex c5, by Avestin (1 mentions)
Histrap ff crude, by GE Healthcare (1 mentions)
U plex kit, by Mesoscale (1 mentions)
Easysep pe selection kit, by STEMCELL (1 mentions)
Ni nta, by GE Healthcare (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Lal chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Sepharose cl 6b, by GE Healthcare (1 mentions)
Superdex 200 10 30 column, by GE Healthcare (1 mentions)
Endothelial growth medium, by Lonza (1 mentions)
Anti phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Gel filtration calibration kit, by Merck Group (1 mentions)
Human aortic endothelial cells haecs, by PromoCell (1 mentions)
31 protocols using hitrap q ff
1
Heterologous Expression and Purification of EfAcpA and EfAcpB
2
Purification of Pdi1p Protein
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The Pdi1p purification protocol was based on that described by Lappi and Ruddock (32 (link)). In brief, cells producing Pdi1p were lysed by incubation at room temperature in the presence of 0.5% (v/v) Triton X-100 and a combination of DNase A (150 U/L culture) and MgCl2 (10 mM) for 30 min.
The resulting lysate was clarified by centrifugation at 20422 × g for 30 min at 4°C and then incubated with Ni-NTA (1 mL/L culture) for 2 h at room temperature. The resin was then collected and washed with 10 mL of 20 mM phosphate, 50 mM NaCl, 10 mM imidazole, pH 7.4 and then 10 mL of 20 mM phosphate, and 50 mM NaCl, pH 7.4. The bound protein was eluted using 1 mL fractions of 20 mM phosphate, 50 mM NaCl, and 50 mM EDTA, pH 7.4, and then buffer exchanged into 20 mM phosphate and 50 mM NaCl, pH 7.4, using a PD10 column.
The resulting mixture was purified using a 5 mL HiTrap Q FF anion exchange column (GE Healthcare) with a nonlinear gradient between 20 mM phosphate, 50 mM NaCl, pH 7.4 (buffer A), and 20 mM phosphate, 1 M NaCl, pH 7.4 (buffer B) (postinjection 12 mL buffer A, 2 mL of 0%–20% buffer B, 20 mL of 20%–75% buffer B, 15 mL of 75%–100% buffer B, and 10 mL of 100% buffer B). The appropriate fractions were then combined and concentrated using a 10 kDa MWCO centrifugal filter before aliquoting at freezing at −20°C.
The resulting lysate was clarified by centrifugation at 20422 × g for 30 min at 4°C and then incubated with Ni-NTA (1 mL/L culture) for 2 h at room temperature. The resin was then collected and washed with 10 mL of 20 mM phosphate, 50 mM NaCl, 10 mM imidazole, pH 7.4 and then 10 mL of 20 mM phosphate, and 50 mM NaCl, pH 7.4. The bound protein was eluted using 1 mL fractions of 20 mM phosphate, 50 mM NaCl, and 50 mM EDTA, pH 7.4, and then buffer exchanged into 20 mM phosphate and 50 mM NaCl, pH 7.4, using a PD10 column.
The resulting mixture was purified using a 5 mL HiTrap Q FF anion exchange column (GE Healthcare) with a nonlinear gradient between 20 mM phosphate, 50 mM NaCl, pH 7.4 (buffer A), and 20 mM phosphate, 1 M NaCl, pH 7.4 (buffer B) (postinjection 12 mL buffer A, 2 mL of 0%–20% buffer B, 20 mL of 20%–75% buffer B, 15 mL of 75%–100% buffer B, and 10 mL of 100% buffer B). The appropriate fractions were then combined and concentrated using a 10 kDa MWCO centrifugal filter before aliquoting at freezing at −20°C.
3
Purification of Ubiquitin and ERD10 Proteins
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Lyophilized ubiquitin (UBQ) was obtained from Sigma Chemical (St. Louis, MO, USA), whereas plant late embryogenesis abundant protein early response to dehydration 10 (ERD10, UniProt P42759) was produced via recombinant expression in Escherichia coli BL21(DE3) Star expression strain and purified as described previously [22 (link)]. In short, purification was carried out through three chromatographic steps: an ion exchange on HiTrap Q FF at pH 9.5 with gradient elution, followed by two gel-filtration steps on Superdex 200 and Superdex 75 columns, on an AKTA Avant (GE Healthcare, Little Chalfont, UK) FPLC system. The purity of the proteins was checked by SDS-PAGE and was found to be at least 98%.
4
Purification of KIF1C Motor Domain
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KIF1C motor domain (residues 1–349) was fused to murine KIF5C residues 329–334 followed by 6×His tag (Nitta et al., 2004 (link)) and purified based on Romberg et al. (1998) (link). After expression in Rosetta 2 cells using 0.5 mM isopropyl B-p-thiogalactopyranside for 16 hr at 16°C, cells were suspended in Buffer B (50 mM NaPO4, pH 7.4, 15 mM imidazole, 250 mM NaCl, 1 mM MgCl2, 25 µM ATP, protease inhibitors) and disrupted by Emulsiflex C-5 (Avestin, Ottawa, ON). Clarified lysate was incubated with Ni-NTA (Qiagen, Santa Clarita, CA) for 1.5 hr at 4°C and after washing with Buffer B was eluted with Buffer B + 200 mM imidazole. The eluate was diluted fivefold with Buffer C (30 mM Hepes, pH 7.4, 1 mM MgCl2, 1 mM EGTA, 25 µM ATP) and applied to HiTrap Q FF (GE Healthcare), washed with Buffer C + 100 mM NaCl, before being eluted by gradient to 500 mM NaCl in Buffer C. Binding of the purified components was assayed in the same manner as in vitro translation-synthesized constructs.
5
Immunology Reagents and Cell Culture Materials
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Antibodies were from Tonbo Biosciences, Biolegend or BD Biosciences. 5-(and−6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) was from Tonbo Biosciences (San Diego, CA). EasySep PE selection kit was from STEMCELL (Vancouver, Canada). Purification columns HisTrap Crude FF and HiTrap Q FF were from GE Healthcare (Piscataway, NJ). U-PLEX kits were from Meso Scale Discovery (Rockville, MD). Double-color ELISPOT kits were from Cellular Technology Limited (Cleveland, OH). N,N-Dimethyldodecylamine N-oxide (LDAO) solution was from Sigma-Aldrich (St. Louis, MO). Limulus Amebocyte Lysate (LAL) endpoint chromogenic kit was from Lonza (Allendale, NJ). All cell culture plastics were TPP brand from MidSci. Recombinant premium-grade GM-CSF was from Miltenyi Biotec (San Diego, CA). RPMI-1640 and fetal bovine serum were from Mediatech (Manassas, VA).
6
Expression and Purification of preS1-Polypeptide
7
Recombinant Expression and Purification of IL-32 Isoforms
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Seven recombinant rIL-32 (α, -β, -γ, -δ, -ϵ, -ζ, and -θ) proteins were expressed in E. coli with 4-h isopropyl β-D-1-thiogalactopyranoside induction at 37°C. rIL-32β, -γ, and -θ were purified with Ni-NTA agarose from Qiagen (Hilden, Germany), and the others were purified with TALON® Magnetic Bead (Takara) using his6-tag at the N-terminus of rIL-32 isoform proteins. Among the affinity-purified proteins, rIL-32β, -γ, and -ϵ were subjected to a high-performance liquid chromatography column from Grace (Stockbridge, GA), and rIL-32α, -δ, -ζ, and -θ were subjected to an anion exchange column (HiTrap Q FF, 1 ml) from GE Healthcare (Chicago, IL, USA). After that, we checked their concentration by silver staining, Bradford assay, and BCA assay. Next, to check the bands of purified rIL-32 isoform proteins, we did western blotting with mouse anti-his6-tag mAb from R&D system (Minneapolis, MN, USA). The rIL-32 proteins were tested with a LAL chromogenic endotoxin quantitation kit from Thermo Fisher (Waltham, MA, USA). The endotoxin level was below 0.5 EU per 1 μg of rIL-32 protein, which is approximately 0.05 ng in 1 μg of rIL-32.
8
Affinity Chromatography Protocol
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Sepharose 6B-CL and Hitrap Q FF were from GE Healthcare. All other chemicals and solvents are analytical or HPLC grade.
9
Endothelial Cell Signaling Pathway Analysis
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Anti-BMP9 antibody (MAB3209), anti-BMP10 antibody (MAB2926), BMP10 prodomain (3956-BP-050), anti-BMP10 propeptide antibody (AF3956), biotinylated anti-BMP10 propeptide antibody (BAF3956), ALK1-Fc (370-AL), human BMP10 GFD (2926-BP-025) were all purchased from R&D Systems, Inc. Anti-phosphoSmad1/5/8 and anti-phosphoSmad1/5 antibody were purchased from Cell Signaling Technology. Anti-ID1 (M085) and anti-ID3 (M100) antibodies were purchased from CalBioreagents (San Mateo, CA). HiTrap Q FF and Superdex 200 10/30 columns were purchased from GE Healthcare. Human pulmonary artery endothelial cells (HPAECs) and endothelial growth medium were purchased from Lonza, UK. Human aortic endothelial cells (HAECs) were purchased from PromoCell. All other tissue culture medium were purchased from Life Technologies. All plasmid and RNA purification kits were purchased from Qiagen. The Gel Filtration Calibration Kit was purchased from Sigma-Aldrich.
10
Purification of His-PPM1D(1-413) Protein
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His-PPM1D(1-413) was expressed in E. coli BL21 (DE3) pLysS cells and purified by affinity purification and gel filtration, as previously described [21 (link)]. The cell pellets were lysed using a French press and then TALON resins (Clontech, Mountain View, CA, USA) with an elution buffer (25 mM HEPES-NaOH pH 6.8, 150 mM imidazole, 200 mM NaCl, 1 mM MgCl2, 10% glycerol, and 0.005% Triton X-100) and were used to purify His-PPM1D(1-413). His-PPM1D(1-413) was further purified using a HiTrap Q FF (GE Healthcare, Chicago, IL, USA) column and eluted with IEX start buffer and IEX elution buffer, followed by Superdex 75 (GE Healthcare Bioscience, Chicago, IL, USA) column with SEC elution buffer (25 mM HEPES-NaOH pH 6.8, 500 mM NaCl, 1 mM MgCl2, 10% glycerol, and 0.005% TritonX-100).
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