A membrane protein detergent screen on AmtB-GFP was performed as previously described16 (link) with minor modifications. Briefly, AmtB-GFP was extracted from purified membranes (described above) with 1-2% (w/v) of the detergent of interest in Buffer B and incubated for one to three hours at room temperature or overnight at 4 °C with gentle agitation. Insoluble material was pelleted by centrifugation at 20,000 g for 25 minutes at 4 °C. The clarified supernatant was loaded onto small Ni-NTA agarose (Qiagen) drip columns (Bio-spin Chromatography columns, Bio-Rad) and washed with several column volumes of Buffer C (200 mM sodium chloride, 20 mM imidazole, 5 mM BME, 50 mM Tris, pH 8.0 at room temperature) supplemented with two times the critical micelle concentration (CMC) of the detergent of interest. AmtB-GFP was eluted with two column volumes of Buffer D (100 mM sodium chloride, 250 mM imidazole, 5 mM BME, 2x CMC detergent of interest, and 50 mM Tris, pH 8.0 at room temperature). Eluted protein was concentrated using a 100 kDa Molecular Weight Cutoff (MWCO) concentrator and buffer exchanged into MS Buffer (2x CMC detergent of interest and 200 mM ammonium acetate, pH 8.0 with ammonium hydroxide) using a centrifugal buffer exchange device (Micro Bio-Spin 6, Bio-Rad).
Bio spin chromatography column
Manufactured by Bio-Rad
Sourced in United States
Bio-Spin Chromatography Columns are disposable, single-use columns designed for small-scale purification of biomolecules. They utilize a packed bed of resin to separate samples based on their size, charge, or affinity interactions. The columns are available in various volumes and resin types to accommodate different sample sizes and purification requirements.
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Lab products found in correlation
Protein assay rapid kit wako, by Fujifilm (3 mentions)
Micro bio spin 6, by Bio-Rad (2 mentions)
Small ni nta agarose, by Qiagen (2 mentions)
Slide a lyzer dialysis cassette, by Thermo Fisher Scientific (2 mentions)
Tau total human elisa kit, by Thermo Fisher Scientific (2 mentions)
Flag peptide, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Chromogenic endotoxin quantitation kit, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Heat inactivated fetal bovine serum, by Atlanta Biologicals (1 mentions)
Qiafilter plasmid maxi kit, by Qiagen (1 mentions)
Doxycycline, by Bio Basic (1 mentions)
Pfuultra 2 dna polymerase, by Agilent Technologies (1 mentions)
Bio gel p 6, by Bio-Rad (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Biobeads sm 2 adsorbent, by Bio-Rad (1 mentions)
Nbd pc, by Avanti Polar Lipids (1 mentions)
Dmem f12, by Wisent (1 mentions)
Deferoxamine maleimide, by Macrocyclics (1 mentions)
16 protocols using bio spin chromatography column
1
Membrane Protein Detergent Screening
2
Purification and Characterization of Recombinant VvhA
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To test the pathophysiological role of VvhA in HT-29 cells, we purified a recombinant protein of VvhA (rVvhA) from V. vulnificus as previously described [2 (link)]. Briefly, the conserved region of the VvhA gene sequence was amplified and then cloned into a pET29a (+) vector (Novagen, Madison, WI, USA) to generate the pKS1201 (Table 1 ). To induce the protein expression, E. coli BL21 (DE3) harboring the pKS1201 was grown in LB media with ampicillin. The cells mixed with Ni-NTA agarose beads (Qiagen, Valencia, CA, USA) were loaded on Bio-Spin Chromatography Columns (Bio-Rad Laboratories, Hercules, CA, USA). The eluted VvhA protein was dialyzed with Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, Hudson, NH, USA) and assessed the level of endotoxin in the purified rVvhA by using Chromogenic Endotoxin Quantitation kit (Thermo Fisher Scientific Inc., Waltham, MA, USA).
3
Purification of His-tagged MsbA Proteins
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His6-tagged MsbA proteins were purified by Ni2+-nitrilotriacetic
acid (NTA) affinity chromatography.15 (link) Approximately
200 μg Ni2+-NTA resin, with binding capacity of up
to 50 mg mL–1 and bead size 45 and 165 μm,
was pre-equilibrated by washing by centrifugation (175 × g, 1 min, 4 °C) five times with five resin volumes
of ultrapure water and twice with five resin volumes of Wash Buffer
A (50 mM K-HEPES (pH 8.0), 0.1 M NaCl, 10% (v/v) glycerol, 0.05% (w/v)
n-dodecyl-β-d -maltoside (DDM), and 20 mM imidazole
(pH 8.0)). Solubilization of target protein was achieved through addition
of membrane vesicles at ∼5 mg mL–1 to solubilization
buffer (50 mM K-HEPES (pH 8.0), 10% (v/v) glycerol, 0.1 M NaCl, and
1% (w/v) DDM). The mixture was gently shaken at 4 °C for 4 h
before transferring to the washed Ni2+-NTA resin to be
again shaken at 4 °C overnight. Resultant resin was transferred
to 2 mL disposable Biospin chromatography columns (Bio-Rad) and washed
with 20 resin volumes of Wash Buffer A and 20 resin volumes of Wash
Buffer B (50 mM K-HEPES (pH 7.0), 0.1 M NaCl, 10% (v/v/) glycerol,
0.05% (w/v) DDM, and 20 mM imidazole (pH 8.0)). Bound protein was
eluted with 400–500 μL of elution buffer (50 mM K-HEPES
(pH 7.0), 0.1 M NaCl, 5% (v/v) glycerol, 0.05% (w/v) DDM, and 150
mM imidazole (pH 8.0)). Purified protein was kept on ice and used
immediately.
acid (NTA) affinity chromatography.15 (link) Approximately
200 μg Ni2+-NTA resin, with binding capacity of up
to 50 mg mL–1 and bead size 45 and 165 μm,
was pre-equilibrated by washing by centrifugation (175 × g, 1 min, 4 °C) five times with five resin volumes
of ultrapure water and twice with five resin volumes of Wash Buffer
A (50 mM K-HEPES (pH 8.0), 0.1 M NaCl, 10% (v/v) glycerol, 0.05% (w/v)
n-dodecyl-β-
(pH 8.0)). Solubilization of target protein was achieved through addition
of membrane vesicles at ∼5 mg mL–1 to solubilization
buffer (50 mM K-HEPES (pH 8.0), 10% (v/v) glycerol, 0.1 M NaCl, and
1% (w/v) DDM). The mixture was gently shaken at 4 °C for 4 h
before transferring to the washed Ni2+-NTA resin to be
again shaken at 4 °C overnight. Resultant resin was transferred
to 2 mL disposable Biospin chromatography columns (Bio-Rad) and washed
with 20 resin volumes of Wash Buffer A and 20 resin volumes of Wash
Buffer B (50 mM K-HEPES (pH 7.0), 0.1 M NaCl, 10% (v/v/) glycerol,
0.05% (w/v) DDM, and 20 mM imidazole (pH 8.0)). Bound protein was
eluted with 400–500 μL of elution buffer (50 mM K-HEPES
(pH 7.0), 0.1 M NaCl, 5% (v/v) glycerol, 0.05% (w/v) DDM, and 150
mM imidazole (pH 8.0)). Purified protein was kept on ice and used
immediately.
4
Affinity-Purification Protocol for Plasma Proteins
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The affinity-purification reactions using both human and mouse plasma were done essentially as described (39 (link)). Briefly, Strep-Tactin Sepharose beads (IBA) were packaged in a Bio-Spin Chromatography Columns (BioRad) and equilibrated in PBS buffer, pH 7.4, for afterwards charged with 10 μg of recombinant TAP-tagged bait, M1 and sfGFP, proteins. TAP-tagged sfGFP was used as a negative control in all experiments. Pooled human, BALB/c mouse or C57BL/6 mouse, plasma was incubated with the protein-charged beads at 37 °C, 800 rpm, 1 h. The beads were washed with 7 ml of ice-cold PBS buffer, pH 7.4, at 4 °C, and the proteins were eluted using 120 μl of 5 mm biotin in PBS buffer, pH 7.4 at room temperature (39 (link)). The samples were precipitated with tri-chloro acetic (TCA), and finally reduced, alkylated and trypsin digested for mass spectrometry.
5
Protein Quantification and Tau Measurement
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A volume of 45μl of the filtered solution was added to 45μl of the guanidine solution and mixed well. The mixed solution (80μl) and the wash buffer (80μl) were placed into 1 ml of the G-10 column set in a Bio-Spin Chromatography Columns (Bio-Rad), in order to remove guanidine hydrochloride (HCl). The Bio-Spin Column was spun for 1 min at 800×g and the filtered solution (∼160μl) was collected. Triplicates of 5μl (total 15μl) were used for the measurements of total protein concentration using the Protein Assay Rapid Kit WAKO (Wako Pure Chemical Industries). The remaining solution was used to measure total tau concentrations using TAU (Total) Human ELISA Kit (Invitrogen).
6
Screening MSP Expression Levels
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To screen the expression level of MSPs, cells were grown in 20 mL culture to OD600 ~0.5 and induced with 0.1 mM IPTG at 16 °C overnight. Bacteria were harvested by centrifugation at 6000 rpm for 10 mins, resuspended in Buffer A and lysed by freeze-thaw plus the addition of 1% Triton at 4 °C overnight. Cell lysates were clarified by centrifugation at 10,000 rpm for 20 mins. The supernatants were incubated with 100 μL His60 Ni Superflow Resins for 20 min with gentle shaking at room temperature. Samples were loaded onto empty micro Bio-Spin chromatography columns (Bio-Rad #7326204), followed by two times wash using buffer B. Proteins were eluted in buffer C and subjected to analysis by SDS-PAGE.
7
Purification and Reconstitution of Membrane Proteins
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POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and NBD-PC were from Avanti Polar Lipids (Alabaster, AL, USA); Bio-Beads SM2 Adsorbent, Bio-Spin chromatography columns and Bio-Gel P-6 were from Bio-Rad (Hercules, CA, USA); Micro-Spin columns with screw caps were from Thermo Scientific (Rockford, IL, USA); 100 × protease inhibitor cocktail was from EMD Millipore (Billerica, MA, USA); DDM was from Anatrace (Maumee, OH, USA); anti-FLAG M2 agarose and 3 × FLAG peptide were from Sigma (St Louis, MO, USA); SNAP-capture resin was from New England Biolabs, Ipswich, MA, USA); PfuUltra II DNA polymerase was from Agilent (Santa Clara, CA, USA); QiaFilter Plasmid Maxi kit was from Qiagen (Valencia, CA, USA); DMEM, 100 × penicillin/streptomycin were from Invitrogen (Grand Island, NY, USA); heat-inactivated fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA); DMEM/F12 was from Wisent (Saint Bruno, QC, Canada); FBS and DPBS from Life Technologies (Waltham, MA, USA); doxycycline from Bio Basic (Markham, ON, Canada); blasticidin and puromycin from Bioshop Canada (Burlington, ON, Canada); Jetprime transfection reagent from Polyplus Transfections (Illkirch, France); and Miniprep kit from Qiagen.
8
Membrane Protein Detergent Screening
9
Deferoxamine-conjugated Recombinant PrP
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To label recPrP with zirconium-89, full length recombinant mouse PrP (23–230) generated in E.coli was first conjugated to deferoxamine-maleimide (Macrocyclics) to produce deferoxamine-conjugated PrP (DFO-recPrP). To ensure that the linker was not conjugated to the lysine-rich heparin binding domain, recPrP was bound to heparin sepharose beads to block the heparin binding sites. To block the binding sites, Heparin Sepharose 6 Fast Flow beads (1 ml) (Healthcare Life Sciences) were loaded into disposable Bio-Spin chromatography columns (Bio-Rad) and packed with 2 ml of equilibration buffer (0.15 M NaCl in 25 mM HEPES, pH 7.4). RecPrP (200 μg) was mixed with 1 ml of equilibration buffer and applied onto the columns. The columns were washed with 3 ml of equilibration buffer. The beads were then transferred to a 1.5 ml eppendorf polypropylene tube and incubated with 200 μl of deferoxamine-maleimide for 24 hours at room temperature with rotation. The bead slurry was transferred to a chromatography column and the recPrP conjugation was stopped after 24 hours by adding 600 μl PBS with 0.1 M glycine (pH 7). The beads were washed with 2 ml equilibration buffer, and the conjugated recPrP (DFO-recPrP) was eluted with 1 ml of elution buffer (0.7 M NaCl, 25 mM HEPES, pH 7.2). DFO-recPrP was concentrated using Zeba spin desalting columns.
10
Affinity Purification of Tagged Proteins
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Various combinations of expression vectors were co-agroinfiltrated into N. benthamiana leaves and samples were harvested at 2.5 days post agroinfiltration and ground in a cooled mortar in GEN buffer (10% [v/v] glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.5% [v/v] Triton X-100 and protease inhibitor cocktail). The supernatants were incubated with anti-Flag M2 affinity agarose (Sigma-Aldrich) in Bio-spin chromatography columns (Bio-rad) for 2 h at 4°C on a rotator, followed by washing with the washing buffer (10% [v/v] glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1mM DTT and 0.1% [v/v] Triton X-100). Elution of purified proteins was as described above [137 (link)].
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