Cold KAP3A purification buffer [50 mM Hepes, Sigma #H3375, 400 mM KCl, 2 mM MgCl2, 0.002% Brij35, 1 mM β-mercaptoethanol, 1 mM imidazole, 50 mM l -arginine, Sigma #A5006, and 50 mM l -glutamate, Sigma #49449 (pH 7.25)] supplemented with protease inhibitors and DNaseI was added to frozen BL21-AI cell pellets expressing a KAP3A construct. Pellets were thawed and cells were lysed by French press at 10.000 psi. The lysate was clarified by centrifugation (184,000g, 45 min, 4°C), and the supernatant was applied to a 5-ml HiTrap column (GE Healthcare) loaded with cobalt(II)-chloride hexahydrate (Sigma). The column was washed with KAP3A purification and buffer containing 10 mM imidazole, and the protein was eluted in KAP3A purification buffer supplemented with 400 mM imidazole. The eluate was concentrated with a Vivaspin concentrator (Sartorius) and supplemented with 8 mM β-mercaptoethanol. Affinity tags were cleaved overnight at 4°C using SUMO protease. KAP3A was concentrated using a Vivaspin concentrator (Sartorius), ultracentrifuged (280,000g, 15 min, 4°C), and gel filtered using a GE Superdex200 10/300 GL column. Peak fractions were pooled, and the protein was aliquoted on ice and snap frozen in liquid nitrogen.
Vivaspin concentrator
Manufactured by Sartorius
Sourced in Germany, France
Vivaspin concentrators are laboratory devices used to concentrate samples through ultrafiltration. They are designed to efficiently concentrate macromolecules such as proteins, enzymes, or nucleic acids from complex solutions. The concentrators utilize a centrifugal force to pass the sample through a semi-permeable membrane, retaining the target molecules while allowing smaller solutes to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Igg sepharose, by GE Healthcare (3 mentions)
Tev protease, by Thermo Fisher Scientific (3 mentions)
2 d clean up kit, by GE Healthcare (3 mentions)
Con a sepharose, by GE Healthcare (2 mentions)
Acrodisc, by Pall Corporation (2 mentions)
Calmodulin affinity resin, by Agilent Technologies (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Jem 1400 transmission electron microscope, by JEOL (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Cobalt 2 chloride hexahydrate, by Merck Group (2 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (2 mentions)
24 well crystallization plates, by Hampton Research (1 mentions)
Acrodisc syringe filter, by Pall Corporation (1 mentions)
Dialysis tubing, by Thermo Fisher Scientific (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Miracloth membrane, by Merck Group (1 mentions)
Brij 35, by Thermo Fisher Scientific (1 mentions)
56 protocols using vivaspin concentrator
1
Purification of Recombinant KAP3A Protein
2
Purification of KIF3A Protein
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Cold KIF3A purification buffer (50 mM NaPi, 400 mM KCl, 2 mM MgCl2 × 6 H2O, 0.001% Brij35, 0.75 mM β-mercaptoethanol, Sigma #M3148, and 0.2 mM ATP) supplemented with protease inhibitors and DNaseI was added to frozen BL21-RIL cell pellets expressing a KIF3A construct. Pellets were thawed and sonicated. The lysate was clarified by centrifugation (184,000g, 45 min, 4°C), and the supernatant was applied to a 5-ml HiTrap column (GE Healthcare) loaded with cobalt(II)-chloride hexahydrate (Sigma #255599). The column was washed with KIF3A purification buffer, and the protein was eluted in KIF3A purification buffer supplemented with 500 mM imidazole (Sigma #I2399). The eluate was concentrated with a Vivaspin concentrator (Sartorius) and supplemented with 8 mM β-mercaptoethanol. Affinity tags were cleaved overnight at 4°C using SUMO protease. In the case of KIF3A-SNAP, the protein was concentrated after labeling using a Vivaspin concentrator (Sartorius), ultracentrifuged (280,000g, 15 min, 4°C), and gel filtered using a GE Superdex200 10/300 GL column. Peak fractions were pooled, and the labeling ratio was determined using NanoDrop. The protein was aliquoted on ice and snap frozen in liquid nitrogen.
3
Purification of INO80-C Complex
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Tandem affinity purification (TAP) was performed to purify the endogenous INO80-C complex from an S. cerevisiae strain encoding TAP-tagged Ino80 (Open Biosystems). Yeast culture was grown in YPD media. Harvested cells were washed once with water. The cells were lysed in buffer E (20 mM HEPES, pH 7.5, 350 mM NaCl, 10 % glycerol, 0.1 % Tween, and 0.5 mM DTT) supplemented with protease inhibitors using a ball mill in the presence of liquid nitrogen. Lysate was clarified at 40,000g at 4 °C for 1 h. Cleared lysate was incubated with IgG-Sepharose (GE Healthcare) at 4 °C for 2 h, and eluted by TEV protease (Invitrogen) cleavage at 4 °C overnight. The TEV elute was then incubated with Calmodulin-Sepharose beads (Agilent Technology) in buffer E plus 2 mM CaCl2 at 4 °C for 2 h, followed by elution in buffer E plus 10 mM EGTA. Purified proteins were concentrated with VIVASPIN concentrators (Sartorius) and dialyzed against buffer E with 1 mM DTT. Subunit composition was confirmed by SDS-PAGE and mass spectrometry.
4
Crystallization of PsyArAT Complexes
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All crystallizations of the reported PsyArAT complexes were performed by the hanging drop vapor-diffusion method at 291 K, using 24-well crystallization plates (Hampton Research, Aliso Viejo, CA, USA). Crystallization drops were set manually at 1 Μl + 1 μL, protein and well solution, respectively. The protein was concentrated to 9–11 mg/mL in 10 mM Tris–HCl buffer, pH 7.5, using Vivaspin concentrators with a 10 kDa cutoff (Sartorius, Göttingen, Germany). Hexagonal crystals of the PsyArAT/DOH complex, with aspartic acid hydroxy-analog (DOH), were obtained in conditions consisting of 2.1 M DL-Malic acid, pH 7.0 and 0.1 M Tris-HCl buffer pH 7.5. Monoclinic crystals of PsyArT/YOH and PsyArAT/FOH were obtained by co-crystallization, using 0.2 M MgNO3, 20% PEG 2000, HEPES buffer, pH 7.5 and a 20-fold molar excess of L-tyrosine and L-phenylalanine hydroxy-analogs (YOH and FOH). C-centered monoclinic crystals of PsyArAT/WOH were obtained by soaking native crystals, grown in the presence of 0.2 M MgNO3 and 20% PEG 3350 in HEPES buffer, pH 7.5, with a 5-fold molar excess of PLP and 10-fold molar excess of 3-indolelactic acid (hydroxy-analog of L-tryptophan–WOH), for 3 h.
5
Glycoprotein Enrichment and Purification
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Ten milliliters of harvested medium was mixed with 2 ml of ConA Sepharose (GE Healthcare Bio-Sciences) and rotated overnight at 4°C. The mixture was then loaded into a column (0.8 cm × 4 cm) (Bio-Rad Laboratories, Hercules, CA, USA) and equilibrated with equilibration buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4). The column was washed with 50 ml of equilibration buffer to remove non-glycosylated and O-glycosylated proteins, and bound N-glycoproteins were eluted with 0.3 M methyl-α-D-glucopyranoside. Absorbance at 280 nm was measured throughout the chromatographic process. Eluted fractions were concentrated 100 times using Vivaspin concentrators (10,000 molecular weight cut-off; Sartorius) and further desalted with 2-D Clean-Up Kits (GE Healthcare Bio-Sciences) and then subjected to 2-DE analysis.
6
Virus Concentration and RNA Extraction
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The processing of the samples was carried out according to Salvador et al. [18 (link)], also taking into account the results described in Salvador et al. [19 (link)]. In summary, in the laboratory, the NanoCeram filters (Argonite; Sanford, FL, USA) with the samples were eluted with 3% beef extract (BD Bios-science; Franklin Lakes, NJ, USA), followed by an organic flocculation process (45 min) and several steps of concentration by centrifugation, resuspension of the sediment with sodium phosphate pH 7.0–7.5, and a final filtration through 0.22 µm pore-size Acrodisc Syringe filters (PALL Corporation; Ann Arbor, MI, USA). The resulting volume (about 32 mL) was aliquoted and kept at -70 °C until the next step. Of the 32 mL, about 20 mL were used for RNA extraction and 12 mL for storage. In the secondary concentration step, the samples were applied in Vivaspin concentrators (Sartorius; Goettingen, Germany) and centrifuged at 8000× g and 4 °C for 6 h. Finally, the concentrates were subjected to RNA extraction and purification with the viral QIAamp RNA Mini kit (Qiagen; Hilden, Germany), according to the manufacturer’s instructions.
7
Purification of Recombinant Proteins in E. coli
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All purified proteins were expressed from the pET28a vector in BL21(DE3) Escherichia coli cells. Expression cultures were grown in LB broth with constant shaking (200–240 rpm) at 37°C for 10 h. The bacteria were incubated in culture medium at 18°C for 36 h to allow bacterial protein expression. Bacterial pellets were resuspended in binding buffer (20 mM Tris-HCl pH 7.9, 500 mM NaCl, 10% glycerol, 7 mM β-mercaptoethanol, 8 M urea, and 25 mM imidazole) containing an EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland), followed by sonication to induce lysis. The supernatant obtained after centrifugation at 27,000 g for 30 min was purified using a HisTrapTM HP column (GE Healthcare, Chicago, Illinois, USA). Next, the eluted proteins were dialyzed in dialysis tubing (Thermo Fisher Scientific) against a storage buffer (50 mM Tris-HCl, 0.5 M NaCl, 10% glycerol and 7 mM β-mercaptoethanol, pH adjusted to 7.4). The proteins were further purified using size exclusion chromatography (Superdex 200 Increase 10/300 GL; GE Healthcare) to remove nonspecific proteins. Vivaspin® concentrators (Sartorius, Göttingen, Germany) were used to increase the concentration of the protein solution, which was subsequently stored at −80°C.
8
Wheat Straw and Seagrass Secretome Analysis
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For each culture condition (wheat straw or seagrass, with or without sea salt), the supernatants collected for three biological replicates as described in the previous section were centrifuged at 8000 rpm for 10 min at 4 °C and were filtered in three steps on glass microfiber filters through 2.7, 1.6 and 0.7 μm (GF/D, A and F filters, respectively, GE Healthcare Life Sciences, WhatmanTM, ThermoFisher Scientific, Madison, WI, USA) and then on 0.4 and 0.2 μm PES membranes (Acrodisc®, Pall Corporation, Saint-Germain-en-Laye, France). Fifteen milliliters of each culture condition were concentrated three times using 10 kDa cut-off Vivaspin concentrators (Sartorius, Les Ulis, France) and then dialyzed against 50 mM sodium acetate buffer pH 5. The protein concentration in the obtained secretome samples was determined by the Bradford method [63 (link)] using bovine serum albumin (BSA) as a standard.
9
Characterization of C. gallica Secretome
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For each culture condition (with or without levofloxacin), supernatants of C. gallica cultures (Day 7) were prepared in triplicate, filtered on a Miracloth membrane (EMD Millipore Corp, Billerica, MA, USA), and centrifuged at 8000× g for 10 min at 4 °C. The resulting supernatants were successively filtered via 2.7, 1.6, and 0.7 μm glass microfiber filters (GD, A, and F, respectively) (GE Healthcare Life Sciences, WhatmanTM, ThermoFisher Scientific, Madison, WI, USA) and 0.4 μm and 0.2 μm PES membranes (Acrodisc®, Pall Corporation, Saint-Germain-en-Laye, France). Then, a 40 mL aliquot of each culture condition was concentrated by ultrafiltration via Vivaspin concentrators (20 mL) at a 10 kDa cut-off (Sartorius, Les Ulis, France), then dialyzed against 50 mM sodium acetate buffer pH 5. The Bradford method was used to determine the protein concentration in the obtained secretome samples with or without levofloxacin, using bovine serum albumin (BSA) as standard [69 (link)].
10
Tandem Affinity Purification of Chromatin Remodelers
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