Chimeric EcAhpC1-186-YFSKHN was concentrated to 8 mg/ml in buffer containing 50 mM Tris-HCl pH 7.5, 200 mM NaCl, using a Millipore spin concentrator with a molecular-mass cutoff of 10 kDa. An initial crystallization attempt was carried out using the recent protocol of EcAhpC (1.8 M ammonium sulfate, 100 mM MES (2- (N-morpholino) ethanesulfonic acid), pH 6.5 and 5% Dioxane)20 (link), and the hanging-drop vapour diffusion method in 24-well VDX plates with sealant at 291 K. Diffraction quality crystals were obtained in the optimized condition of 1.6 M ammonium sulfate, 100 mM MES pH 6.5, 5% Dioxane and a protein concentration of 4.5 mg/ml, yielding rod shaped crystals of 0.3 mm × 0.2 mm × 0.1 mm. Reduced EcAhpC1-186-YFSKHN crystals were grown by soaking the oxidized crystals with 1 mM Tris(2-carboxyethyl) phosphine (TCEP) for 1–3 min. There was no cracking or disintegration of crystals observed during the soaking.
Spin concentrator
Manufactured by Merck Group
The Spin concentrator is a laboratory device used to concentrate samples through centrifugation. It reduces the volume of liquid samples by removing solvents or other volatile components, allowing for increased sample concentration.
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Lab products found in correlation
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Microfluidizer, by Microfluidics (1 mentions)
Superdex s200, by Cytiva (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Pierce rna 3 end biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Histrap hp column, by GE Healthcare (1 mentions)
Bugbuster, by Merck Group (1 mentions)
Superdex 200 16 60 gel filtration column, by GE Healthcare (1 mentions)
Econo pac column, by Bio-Rad (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Superdex 75 column, by GE Healthcare (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (1 mentions)
9 protocols using spin concentrator
1
Crystallization of Chimeric EcAhpC1-186-YFSKHN
2
Purification of Recombinant COX-2 Proteins
3
Sulfo-Cyanine-7.5 Labeling of IgG4 Antibody
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An IgG4 isotype antibody was labeled with Sulfo‐Cyanine‐7.5 dye using NHS ester chemistry (66320, Lumiprobe). The antibody was prepared at ≈10 mg mL−1 in 90 mm carbonate, 9 mm phosphate buffer with 125 mm sodium chloride at pH 8.3. The dye was dissolved in a one‐tenth volume of carbonate buffer immediately before adding to the antibody at a 10 equivalent excess and incubated at 25 °C for 4 h. The labeled antibody was isolated from the excess dye on a size exclusion chromatography column (Superdex S200, Cytiva) with a mobile phase of 1× PBS, pH 7.2 at 1 mL min−1. The degree of labeling was determined using MALDI‐MS to be ≈4.5 dyes per antibody. The antibody was concentrated to ≈30 mg mL−1 using a 15 mL spin concentrator (Millipore) with a 100 kDa MWCO membrane. Samples of labeled and unlabeled antibody (5–10 µg) were run on an HPLC (Agilent 1260 Infinity II) using an Agilent AdvanceBio SEC 300Å 2.7 mm column (PL1580‐3301, Agilent) at 1 mL min−1 in a mobile phase of 1× PBS; pH 7.2 (20012‐027, GIBCO) with peaks detected by absorbance at 214 nm. The total run time was 7 min.
4
Preparation and Labeling of tRNA Transcripts
5
Deuterium Exchange of Hydrogenase Enzyme
6
Structural Characterization of Ran-RanBP1-CRM1 Complex
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Untagged RanY197A, GST-NESMVM (or mutants), GST-yRanBP1, and GST-yCRM1ΔH9 were expressed in E. coli separately and mixed, sonicated in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl,10% glycerol, 2 mM DTT, 5 mM MgCl2 and 1 mM PMSF). The complex was purified by GSH beads, cleaved off from beads by incubating the TEV enzyme overnight. The complex was further purified by gel filtration Superdex200 (GE Healthcare) column in buffer D (10 mM Tris 7.5, 100 mM NaCl, 5 mM Mg(OAc)2, 0.1 mM GTP, 2 mM BME). The complex was concentrated to 6 mg/mL using a Millipore spin concentrator (M.W. cutoff 10, 000). Crystals of different complexes were grown at room temperature by hanging drop vapor diffusion against 0.1 M Bis-Tris (pH 6.6), 0.2 M NH4NO3, and 18% PEG3350. Crystallization condition supplemented with glycerol (12% v/v) was used the as the cryoprotectant. X-ray diffraction data were collected at Shanghai Synchrotron Radiation Facility (SSRF) beamline BL17U1 and BL19U1.34 (link) Coordinates of yCRM1-hRan-yRanBP1 (PDB code: 4HAT) were used as the search model and refined with rigid body refinement briefly then restrained refinement using the program Refmac5.35 (link) Translation/Libration/Screw (TLS) refinement36 (link) was used in the refinement process. The data collection and refinement statistics are provided in Table S1
7
Purification of Tau P301S Protein
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Tau P301S_10×his-tag_avi-tag was overexpressed in BL21(DE3) bacteria. Cells were lysed using BugBuster (Millipore), and clarified lysate was applied to a 5-mL HisTrapHP column (GE Healthcare) in 2× PBS. Tau was eluted using a 0- to 500-mM imidazole gradient. Peak fractions were pooled and further purified in 2× PBS using a Superdex 200 16/60 gel filtration column (GE Healthcare). Pooled fractions were then concentrated to approximately 8 mg/mL using a spin concentrator (Millipore). Final protein concentration was determined by Nanodrop analysis.
8
Two-Step Purification of C Proteins
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We purified both C proteins in two steps: Nickel Immobilized Affinity Chromatography (IMAC) followed by size-exclusion chromatography (SEC). Pellets were thawed and homogenized (Constant Systems homogenizer) at 25 kpi at 4°C. The lysate was cleared at 50,000 g for 30 minutes before batch incubation of the supernatant (i.e. the soluble fraction of bacteria) on Ni-NTA sepharose FF resin (Qiagen) for 2 hrs at 4°C. The material was collected in an Econo-Pac column (Biorad) and washed in 100 Column Volumes (CV) of lysis buffer. Elution was done in 0.5 CV fractions with lysis buffer containing 500 mM Imidazole. Fractions containing protein were pooled and loaded onto a preparative Superdex 75 (GE Healthcare Life Sciences) size exclusion column pre-equilibrated in 20 mM Tris 150 mM NaCl, 1 mM EDTA, pH 7.5. Peak fractions were pooled and concentrated using 15 ml spin concentrators (Millipore).
9
Purification of SpoVG transcription factor
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spoVG I was amplified from the L. monocytogenes chromosome by using primers TB254/TB255, digested, ligated into pET20b, and transformed into BL21 cells containing pLysS. Bacterial cultures (1.4-liter cultures) were grown with shaking at 37°C until the OD600 reached 0.40, and spoVG expression was induced with 1 mM isopropyl-β-d -thiogalactopyranoside (Sigma) for 2 h. Bacteria were then collected by centrifugation, flash-frozen, and lysed by sonication in buffer A [300 mM NaCl, 50 mM Tris, 25 mM imidazole, 0.5 mM Tris(2-carboxyethyl)phosphine, 20% glycerol; pH 8.0]. Cell wall debris was removed by centrifugation, and the resulting lysate was passed over Ni-nitrilotriacetic acid (NTA) affinity resin (Thermo). The resin was washed with a minimum of 40 ml buffer A followed by elution with increasing concentrations of imidazole (50, 75, 100, 125, and 300 mM). Elutions 3 to 5 were pooled and dialyzed overnight into buffer B (1 mM dithiothreitol [DTT], 50 mM Tris-HCl, 25 mM KCl, 2 mM MgCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride; pH 8.0). Proteins were then separated by size exclusion chromatography using a Superdex 75 column (GE). Low-molecular-weight fractions (fractions 17 to 23) were pooled, concentrated using spin concentrators (Millipore), and evaluated in EMSAs. Protein concentrations were determined via the Bradford assay (Bio-Rad).
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