Ub(1-75)-MesNa was prepared as described previously61 (link). 0.7 g of propargylamine hydrochloride (Aldrich, P5091) were dissolved in 7 mL of buffer D (20 mM Hepes, 50 mM sodium acetate, pH 6.5, 75 mM sodium chloride), supplemented with 490 µL of 4 M sodium hydroxide, and added to 6 mL of 600 µM Ub(1-75)-MesNa in buffer D. The final pH of the reaction mixture was between 8.0 and 8.5. Following incubation at room temperature for 3 h, completion of the reaction was observed by intact protein mass spectrometry (expected mass: 8,544.8 Da, mass found: 8,544.1 Da). The reaction mixture was then concentrated to 2.5 mL in a spin concentrator (3 kDa MWCO, Amicon Ultra) for size-exclusion chromatography (HiLoad 16/600 Superdex 75 pg, GE Healthcare) in buffer D to yield ubiquitin(1-75)-propargylamine suicide probe (Ub-PA).
Hiload 16 600 superdex 75 pg
Manufactured by GE Healthcare
Sourced in United States, Germany
The HiLoad 16/600 Superdex 75 pg is a size exclusion chromatography column designed for the purification of proteins, peptides, and other biomolecules. It features a packed bed height of 600 mm and an internal diameter of 16 mm, making it suitable for medium-scale purification applications.
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Lab products found in correlation
Superdex 200 pg 10 300 gl column, by GE Healthcare (3 mentions)
Talon metal affinity resin, by Takara Bio (3 mentions)
Xjb de3 autolysis, by Zymo Research (3 mentions)
Propargylamine hydrochloride, by Merck Group (2 mentions)
Blue dextran, by Merck Group (2 mentions)
Aldolase, by Merck Group (2 mentions)
Carbonic anhydrase, by Merck Group (2 mentions)
Conalbumin, by Merck Group (2 mentions)
Kta purifier, by Cytiva (2 mentions)
Amersham protran 0.45 μm nc, by GE Healthcare (2 mentions)
Penta his antibody horseradish peroxidase hrp conjugate, by Qiagen (2 mentions)
Rnase a, by Merck Group (2 mentions)
Mono q 5 50 gl column, by GE Healthcare (2 mentions)
Anti h1, by Santa Cruz Biotechnology (2 mentions)
Anti ack, by Cell Signaling Technology (2 mentions)
Histrap ff column, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Histrap chelating hp column, by GE Healthcare (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
34 protocols using hiload 16 600 superdex 75 pg
1
Synthesis of Ub(1-75)-Propargylamine Suicide Probe
2
Copper Binding Protein Purification
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The purified proteins were exchanged into buffer (20 mM Tris-MES, pH 8.0) by centrifugal ultrafiltration (Millipore) and incubated for 30 mins with CuSO4 and reduced GSH (molar ratio 1:5:10; protein: CuSO4: GSH). In order to remove the excess Cu from the mixture, the incubated protein sample was applied to a SEC column (HiLoad 16/600 Superdex 75 pg, GE Healthcare). The presence of Cu(I) in the peak fractions was analyzed colorimetrically using the ligand bathocuproinedisulfonic acid (Bcs) and those protein fractions containing Cu were pooled and concentrated by centrifugal ultrafiltration. These analyses indicated excellent resolution between the Cu(I)-protein and excess Cu-GSH peaks (data not shown). The Cu(I):protein stoichiometries of the protein samples prepared in this manner were confirmed by a colorimetric assay using Bcs10 (link).
3
Recombinant Protein Expression and Purification in E. coli
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All proteins were expressed in XJb(DE3) Autolysis (Zymo Research, Irvine, CA, USA) E. coli strain. Cells were grown in LB or M9 media supplemented with 100 μg·mL−1 ampicillin (full-length wtBlc protein) or 100 μg·mL−1 ampicillin and 50 μg·mL−1 kanamycin (wtBlc-split-Zip and wtBlc-split proteins) at 37 °C. Expression was induced by addition of 0.04% L-arabinose (full-length wtBlc protein) or 0.2% L-arabinose and 10 μM IPTG (wtBlc-split-Zip and wtBlc-split proteins) at 0.8 OD. Cells were harvested after 3 h of expression at 37 °C if grown in LB or after overnight expression if grown in M9 and were resuspended in PBS buffer, pH 7.4. Suspensions were frozen at −80 °C and thawed at room temperature three times. DNA was destroyed by short sonication, and the lysates were centrifuged to obtain cell-free extracts. The proteins were first purified using gravity flow columns with TALON metal affinity resin (Clontech, Mountain View, CA, USA) and further purified by size-exclusion chromatography on a HiLoad 16/600 Superdex 75 pg or Superdex 200 pg 10/300 GL column (GE Healthcare, Marlborough, MA, USA) pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0.
4
Recombinant SARS-CoV-2 Spike Protein Purification
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The cloning, expression, and purification of the N-terminal region of the Spike protein were previously described [8 (link)]. Methodology details are in a supplementary file (Figure S7) . The SARS-CoV-2 Spike DNA fragment corresponding to the residues from 16 to 165 (rSpike) was amplified by PCR using a SARS-CoV-2 cDNA. The purified PCR product was digested using AnzaTM restriction enzymes NheI and BamHI (Thermo Fisher Scientific, Waltham, MA, USA), and the same pair of enzymes were used to digest the expression vector pET-28a. The digested and purified plasmid was used to ligate the rSpike DNA fragment. Digestion tests confirmed the positive clones. This cloning results in a fusion of seven histidine tags at the N-terminal portion of the protein. rSpike was purified by expression of the protein in Escherichia coli strain BL21(DE3) or BL21 StarTM (DE3), followed by two steps of purification using HisTrap Chelating HP column (GE Healthcare Life Sciences, Chicago, IL, USA) and HiLoad 16/600 Superdex 75 pg (GE Healthcare Life Sciences) size exclusion chromatography. The purified protein (7 M urea, 50 mM MOPS, 200 mM NaCl, 1 mM EDTA, pH 7.0) was concentrated using Amicon Ultra-15 Centrifugal filters (Merck Millipore, Milwaukee, WI, USA) with a 3 kDa membrane cut-off.
5
Purification of Ucc1-Skp1 Complex
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To purify the Ucc1-Skp1(FL) series for assay, frozen cell pellets were thawed and resuspended in lysis buffer. Subsequently, the cells were lysed by sonication, and cell debris was removed by centrifugation. The supernatant was subjected to Ni-NTA resin affinity column chromatography. Unbound proteins were washed out with Ni-NTA column washing buffer, and then the bound protein was eluted from the column using Ni-NTA column elution buffer. The eluates were dialyzed overnight at 4°C against size exclusion column buffer B [40 mM tris (pH 7.5), 60 mM NaCl, and 10% glycerol]. The HisSUMO1-tag was cleaved from the complex by treatment with a HisSUMO1-tagged Ulp1 protease (in-house) after dialysis. To separate the cleaved HisSUMO1-tag and the Ucc1-Skp1 complex, proteins were subjected to size exclusion column chromatography (HiLoad 16/600 Superdex 75 pg, GE HealthCare) using an FPLC system. The column was equilibrated and eluted with size exclusion column buffer B. The eluates were dialyzed overnight at 4°C against dialysis buffer and then concentrated to ~5 mg/ml using a centrifugal concentrator.
6
Synthesis of Ub(1-75)-Propargylamine Suicide Probe
7
Soluble eAds Protein Purification
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The soluble eAds were expressed and purified as previously described 28 (link). The proteins were purified by Ni-NTA column. Eluted proteins were purified by the size-exclusion column (GE HiLoad 16/600 Superdex 75pg) using AKTA Avant 150 (GE Healthcare). A 4ml sample was loaded into the size-exclusion column and equilibrated with equilibrating buffer (1mM KH2PO4, 155mM Nacl, 3mM Na2HPO4-7H2O, PH 7.4). The column was washed continuously by using the buffer (1mM KH2PO4, 155mM Nacl, 3mM Na2HPO4-7H2O, PH 7.4) at the flow rate of 1ml/min, and the fraction of the major area was collected.
8
Preparation of Recombinant Fluorescent Proteins
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The preparation of 4B08 antibody was previously described16 (link). For the preparation of GFP-NT, GFP-CT, and GFP-C0, Escherichia coli strain BL21(DE3) carrying the expression vector of protein-fused peptides was grown overnight at 28 °C, 170 rpm in LB plate medium. The cells were diluted into 100 mL of LB medium and cultured at 37 °C and 140 rpm until the OD600 reached a value of 0.2. At that point isopropyl β-d -1-thiogalactopyranoside was added to the cell culture to a final concentration of 0.5 mM and the cell culture was left overnight at 37 °C. Cells were harvested by centrifugation at 7,000 × g for 20 min at 4 °C. The cell-pellet was resuspended in 40 mL of TRIS buffer (20 mM Tris, 500 mM NaCl, pH 8.0) supplemented with 5 mM imidazole, and the cells lysed with an ultrasonic cell-disrupting UD-201 instrument (TOMY, Japan). The cell lysate was subsequently centrifuged at 4 °C, 40,000 × g for 30 min. The supernatant was collected, and further purification was conducted by size exclusion chromatography (AKTA purifier with Hiload 16/600 superdex 75 pg, GE healthcare) with TRIS buffer at a flow rate of 1.0 mL/min at 4 °C.
9
Recombinant Transthyretin Protein Purification
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Recombinant wt-TTR- and L55P-TTR were expressed and purified according to Mangione et al. [46 (link)]. Lyophilized TTR was dissolved at 1.6 mM in 30 mM sodium phosphate buffer, and pH 7.0. M-TTR was purified as previously reported [12 (link)]. In brief, the cleared lysate was loaded on a Q-Sepharose High Performance resin (GE Healthcare) and eluted with a sodium chloride gradient. Then, the protein was further purified by gel filtration with a HiLoad 16/600 Superdex 75 pg (GE). The eluted protein was dialyzed against 20 mM potassium phosphate, 150 mM NaCl buffer at pH 7.3 enriched with 2.0 mM DTT.
10
Purification and Characterization of Mirolase
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