Recombinant α-Synuclein was purified from bacteria BL21(DE3) harboring pRK172-human α-synuclein using a Q Sepharose column (17051010, Cytiva, Tokyo, Japan).79 (link) Recombinant Tau was purified from bacteria BL21(DE3) harboring pRK172-4R2N Tau using an SP Sepharose column (17072910, Cytiva).81 (link) The third instar larvae were dissected in Schneider medium (cat. no. 21720024, Gibco, Thermo Fisher Scientific, Waltham, MA) with 10% fetal bovine serum. The motor neuron axon was sucked into a glass electrode and stimulated with 5 V, 1 Hz for 10 min in Schneider medium with 10 μM α-Synuclein or Tau. After stimulation, dissected larvae were incubated for 30 min in Schneider medium with 10 μM α-Synuclein or Tau and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS).
Q sepharose column
Manufactured by Cytiva
Sourced in Japan
The Q Sepharose column is a chromatography column used for the purification of biomolecules. It is a strong anion exchanger that can be used to separate and purify a variety of negatively charged molecules, such as proteins, nucleic acids, and other biomolecules. The column is packed with Q Sepharose resin, which provides a high-capacity and high-resolution separation platform.
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Lab products found in correlation
Sp sepharose column, by Cytiva (1 mentions)
Pierce immobilized tcep disulfide reducing gel, by Thermo Fisher Scientific (1 mentions)
Ez link maleimide peg11 biotin, by Thermo Fisher Scientific (1 mentions)
Histrap column, by Cytiva (1 mentions)
Ni sepharose 6 fast flow column, by Cytiva (1 mentions)
Mab select sure protein a, by GE Healthcare (1 mentions)
Heparin column, by Cytiva (1 mentions)
Superdex 200 size exclusion column, by Cytiva (1 mentions)
Amicon ultra 15 centrifugal filter device, by Merck Group (1 mentions)
Bca kit, by Tiangen Biotech (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Human ace2 duoset elisa kit, by R&D Systems (1 mentions)
Hitrap protein g, by Cytiva (1 mentions)
Hiload 16 600 superdex, by Cytiva (1 mentions)
Pngase a, by New England Biolabs (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
10 protocols using q sepharose column
1
Purification and Stimulation of α-Synuclein and Tau
2
Purified ptfV Cysteine Variant Labeling
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Purified ptfV cysteine variants were reduced with Pierce Immobilized TCEP disulfide reducing gel (Thermo Fisher Scientific) at room temperature for 45 minutes, as per the manufacturer’s instructions. Reduced samples were separated from the TCEP gel by centrifugation using a spin cup paper filter and then labeled with 20-fold molar excess of maleimide-PEG40K (Merck) or EZ-Link maleimide-PEG11-biotin (922.09 Da; Thermo Fisher Scientific) at 4°C for 2 hours. Maleimide-PEG40K–labeled samples were used to estimate labeling efficiency by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE; Table 1 ; supplemental Figure 1). Maleimide-PEG11-biotin–labeled samples were applied to a Q Sepharose column (Cytiva; 1 mL) followed by dialysis to remove excess labeling reagent. Peak fractions containing both the mixture of labeled and unlabeled ptfV were pooled and dialyzed into a buffer of 100 mM of potassium phosphate and 150 mM of NaCl (pH 7.2) overnight at 4°C. Dialyzed samples were applied to a column packed with streptavidin mutein resin (Roche) to separate biotinylated from unbiotinylated protein, as per the manufacturer’s instructions (supplemental Figure 2). Peak fractions containing biotinylated ptfV were concentrated and dialyzed overnight into a buffer of 20 mM of Tris (pH 7.4), 150 mM of NaCl, and 2 mM of calcium chloride at 4°C.
3
Reconstitution and Purification of γ-Complex
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To reconstitute the γ-complex, we combined purified His-δ′, δ, and γ proteins in a stoichiometric ratio of 0.75:1:3, and the protein mixture was then dialyzed overnight into Buffer Ni-A. We then loaded the protein onto a 5 ml HisTrap column (Cytiva), then eluted it with Buffer Ni-B. Fractions containing the assembled complex were combined with Prescission Protease and were dialyzed overnight into Buffer Ni-A. The γ-complex was then passed over a 5 ml HisTrap column, and the flowthrough was collected and dialyzed into Buffer Q-A. The dialyzed protein was then loaded onto a 5 ml Q-sepharose column (Cytiva) and eluted with a 10-column volume gradient of 50 mM Tris pH 7.5, 500 mM NaCl, 10% glycerol (v/v), and 2 mM DTT (Buffer Q-B). Fractions containing the intact γ-complex were then pooled and dialyzed into storage buffer. We then concentrated the purified γ-complex to ∼2.5 mg/ml using an Amicon Ultra-15 centrifugal filter with a 30,000 kDa molecular weight cutoff. Aliquots were flash-frozen in liquid nitrogen and stored at −80 °C.
4
Purification and Production of ActRIIA-Fc, ActRIIB-Fc, and Chimeric Receptors
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ActRIIA-Fc was purchased from R&D (Cat. No. 340-RC2-100, Minneapolis, MN). ActRIIB-Fc was expressed and purified from Chinese hamster ovary (CHO) cells as previously described (Sako et al., 2010 (link); Cadena et al., 2010 (link)). Briefly, ActRIIB-Fc was isolated using affinity chromatography with Mab Select Sure Protein A (GE Healthcare, Waukesha, WI), followed by dialysis into 10 mM Tris, 137 mM NaCl, and 2.7 mM KCl, pH 7.2. ActRIIB-ALK7-Fc and ActRIIB-ALK4-Fc were designed and expressed as previously described in CHO DUKX cells through the coexpression of two plasmids, each containing a receptor ECD (ActRIIB or ALK4) fused to a modified human IgG1 Fc domain (Li et al., 2021 (link); Kumar et al., 2021 (link)). Purification was performed through protein A MabSelect SuRe chromatography (Cytiva), then eluted with glycine at low pH. The resulting sample was further purified over a Ni Sepharose 6 fast flow column (Cytiva) followed by an imidazole elution gradient, an ActRIIB affinity column and ultimately, a Q Sepharose column (Cytiva). ALK7-Fc production was performed as described previously (Kumar et al., 2021 (link)).
5
Purification of Recombinant Prothrombin
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A quick change lightning site-directed mutagenesis kit (Agilent Technologies) was used to introduce substitutions into a plasmid expressing the human prothrombin gene carrying a C-terminal HPC-4 tag. Transfection of baby hamster kidney cells and selection of stably expressing clones were done as described for protein C (39 (link)). Prothrombin secreted in media (6–8 L) collected from CellSTACK culture factories with ten chambers (Corning) was initially purified by immunoaffinity chromatography (40 (link)). After diluting the [NaCl] < 40 mM, prothrombin was loaded onto a 1 ml Q-sepharose column (Cytiva) attached to the bottom of a 1 ml Heparin column (Cytiva) equilibrated with 20 mM Tris, pH 7.5, 40 mM NaCl, and 10 mM EDTA. The Heparin column was detached and prothrombin was eluted from the Q-sepharose column using 0.04–1 M NaCl gradient. Protein purity was further refined on a superdex 200 size-exclusion column (Cytiva).
6
Purification of Antibiotic Resistance Protein RAD-1
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E. coli BL21 (DE3) harbouring pET24a-blaRAD-1-no-sp was grown to an OD600 of 0.6 at 37°C in 1 L of LB broth. Induction, crude protein extract preparation, and ion-exchange purification of RAD-1 were performed as described previously with a few modifications.28 (link) Briefly, the growing culture was induced by 0.2 mM IPTG (Sigma–Aldrich) overnight at 25°C. The cells were harvested by centrifugation and resuspended in buffer A (50 mM Tris-H2SO4, pH 7.0). After disruption by sonication, the supernatant was separated by centrifugation at 12 000g for 40 min at 4°C and filtrated by 0.22 μm membranes. Subsequently, the cleared lysate was loaded onto a pre-equilibrated Q-Sepharose column (Cytiva) in buffer A. The enzyme recovered in the flowthrough was dialysed overnight at 4°C against buffer B (50 mM Tris-H2SO4, pH 8.0). The enzyme fraction was then loaded onto a CM cation-exchange column (Cytiva), pre-equilibrated with the same buffer, and eluted with a linear K2SO4 gradient (0 to 500 mM) to prevent any inhibition by NaCl. In purification processes, the enzyme activity was assessed by nitrocefin. Fractions were analysed by SDS-PAGE. The RAD-1 was collected and dialysed against buffer A. Finally, the protein was concentrated using a 10 kDa Amicon® Ultra-15 centrifugal filter device (Millipore), and the concentration was determined using the BCA kit (Tiangen).
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E. coli BL21 (DE3) harbouring pET24a-blaRAD-1-no-sp was grown to an OD600 of 0.6 at 37°C in 1 L of LB broth. Induction, crude protein extract preparation, and ion-exchange purification of RAD-1 were performed as described previously with a few modifications.28 (link) Briefly, the growing culture was induced by 0.2 mM IPTG (Sigma–Aldrich) overnight at 25°C. The cells were harvested by centrifugation and resuspended in buffer A (50 mM Tris-H2SO4, pH 7.0). After disruption by sonication, the supernatant was separated by centrifugation at 12 000
7
Purification and Characterization of ACE2 Proteins
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ACE2 and ACE2 lacking the membrane-binding domain (ACE2s) were produced by growing strains sCX00037 and sCX00148 respectively, in FM02 medium. Culture supernatants were collected by centrifugation at 3000 x g after overnight incubation at 30°C. The harvested culture supernatants were then dialyzed overnight against 2 liters of 25 mM Tris-HCl (pH 8). Dialyzed supernatants were then loaded to a Q Sepharose column (Cytiva, Marlborough, MA) equilibrated with 25 mM Tris-HCl (pH 8). Proteins were eluted with a linear gradient to 1 M NaCl. Elution fractions were tested for presence of ACE2(s) by western blot and ELISA (Human ACE-2 DuoSet ELISA kit, R&D Systems, Minneapolis, MN). Fractions with the highest concentration ACE2(s) were then tested for ACE2 activity using a commercial activity assay (BioVision, Milipitas, CA). Non-specific protease activity was determined by adding ACE2 inhibitor to samples before testing their activities. The manufacturer’s instructions were followed with the exception that the provided positive control was diluted to similar activity levels.
8
Affinity Purification of Antibodies
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Antibodies in the cell culture harvest were purified by capturing on a HiTrap protein G (Cytiva), followed by washing with PBS and elution with 0.1 M Glycine pH 2.7. After dialysis against 20 mM Tris pH 7.5, the sample was passed through a Q-sepharose column (Cytiva) and equilibrated with the same buffer. The flow-through was concentrated to < 5 mL and purified on a HiLoad® 16/600 Superdex® (Cytiva) using PBS as eluent. Fractions were analyzed by SDS-PAGE, SEC, and LC-MS. Selection for pooling was made to minimize aggregates, incorrectly paired molecules, and free LC. After purification, a certain amount of Hole dimer (double Hole HC with typical Fc length) was still present in the used Adu H310A -8D3 and B12 H310A -8D3 batches (Figure S3 and S4; t R = 15.2/16.3; ~ 20%).
9
Refolding and Purification of Chemokines
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Inclusion bodies were solubilized for 1 h at room temperature in buffer C (100 mM Tris-HCl, pH 8.0) supplemented with 6 M Urea, 100 mM NaCl, 0.5 mM EDTA and 5 mM DTT. Refolding was achieved by drop-wise dilution into a volume 100 times that of the Urea solution of buffer C containing 0.5 mM EDTA, 0.2 mM oxidized glutathione and 1 mM reduced glutathione. The respective refolding solution was stirred overnight at 4°C and loaded onto a 10 mL Q-sepharose column (Amersham Biosciences) pre-equilibrated in buffer B. A linear gradient elution was performed over 100 mL from 0 to 1 M NaCl in buffer B and the fractions containing the refolded chemokines were concentrated with Centricon cell and further purified over a Superdex 200 gel filtration column equilibrated with buffer B containing 150 mM NaCl.
10
Extraction and Purification of Recombinant CBM3GH5 Enzyme
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Extraction in nondenaturing condition was performed from 5 g FW CBM3GH5-HA-VAC#4-11 leaf material, by a modified buffer (10 mM Tris-HCl pH 7.5, 0.4% Tween 20) at the optimized ratio [2 mL extraction buffer: 1 g FW leaf]. After 1 h incubation at 70 °C, the sample was centrifuged at 14,000× g 10 min to promote the precipitation of thermal-denaturated protein [40 (link)]. Supernatant was loaded on a Q-sepharose column (Amersham) equilibrated with 20 mM Tris-HCl pH 7.5. Elution was performed by using a stepwise NaCl gradient (from 0 to 1 M NaCl, with 0.1 M increments). Fractions eluted from the Q-sepharose column were tested by enzymatic assay and analyzed by SDS-PAGE for determination of enzyme concentration using different amount of BSA as calibration standard. Protein concentration was assessed using the Quantity-One software (Biorad). The specific activity of the enzyme (Units mg enzyme−1) was used for determining the amount of recombinant enzyme in CBM3GH5HAVAC#4-11 plants. Deglycosylation of purified CBM3GH5 isoforms was performed by using PNGase A according to the manufacturer’s instructions (P0707, New England Biolabs).
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