Biotinylated RBD proteins from ancestral SARS-CoV-2, SARS-CoV-2 Alpha, Delta, Beta, Gamma, bat CoV RaTG13, Pangolin CoV GX-P5L and SARS-CoV-1 were custom-made by Genscript. Biotinylated SARS-CoV-2 Omicron RBD was purchased from Acrobiosystems. Biotinylated RBDs from SARS-CoV-2 Delta plus, Mu and Lambda, bat CoVs WIV1, Rs2018B, LYRa11 and RsSHC014 were produced in-house. Briefly, the RBD coding sequences were cloned into pcDNA3.1 vector with SARS-CoV-2 signal peptide (amino acid 1-14) at the N-terminus and 10x his-tag followed by AviTag at the C-terminus. After transfection of expression plasmid into HEK293T cells using FuGENE6 in Opti-MEM media, expressed proteins were harvested at day 3 or day 6 post-transfection, respectively. RBD proteins were purified using Ni Sepharose (GE Healthcare) and desalted using Amicon Ultra-4, 10K MW (Merck). Enzymatic biotinylation of AviTag was performed using BirA Protein-Biotin ligase kit (Avidity) according to the manufacturer’s instructions. Excessive biotin was removed by Amicon Ultra-4, 10 MW (Merck). Protein concentration was determined by Nanodrop (DeNovix).
Amicon ultra 4
Manufactured by Merck Group
Sourced in United States, Germany, Ireland, Italy, United Kingdom
The Amicon Ultra-4 is a centrifugal filter device designed for the concentration and desalting of protein and other macromolecular solutions. It is capable of processing sample volumes up to 4 milliliters.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by GE Healthcare (4 mentions)
Gelatin, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Hitrap, by Cytiva (3 mentions)
Pkh26 red fluorescent labelling kit, by Merck Group (3 mentions)
Amicon ultra 15, by Merck Group (3 mentions)
Expi293 cell, by Thermo Fisher Scientific (2 mentions)
Ni nta agarose resin, by Thermo Fisher Scientific (2 mentions)
Gelatin zymography kit, by Cosmo Bio (2 mentions)
Hiprep, by Cytiva (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Pd mini trap, by Cytiva (2 mentions)
Sepharose cl 4b, by Merck Group (2 mentions)
Stainless steel extruder, by Avanti Polar Lipids (2 mentions)
Fbs media supplement, by System Biosciences (2 mentions)
Superdex 200, by GE Healthcare (2 mentions)
Sep pak 3 cc c18 cartridge, by Waters Corporation (2 mentions)
Superdex 200 10 300, by GE Healthcare (2 mentions)
Trypsin, by Promega (2 mentions)
263 protocols using amicon ultra 4
1
Production and Biotinylation of SARS-CoV-2 RBDs
2
Radiolabeling of αFAP Targeting Molecules
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To modify αFAP TMs with the chelator NODAGA the first step involved the dilution of the purified αFAP TMs in modification buffer (0.1 M Na2B4O7*10 H2O, pH 9). Thereafter, p-NCS-benzyl-NODA-GA (CheMatech) was added in a molar ratio of 20:1 (chelator to TM) and incubated for 4 h at 30 °C. Non-bound p-NCS-benzyl-NODA-GA was removed by spin filtration using Millipore AmiconUltra-4 (MWCO 10,000 for αFAP-scFv TM or 50,000 for αFAP-IgG4 TM) and PBS. Once modification was completed, 1 nmol NODAGA-modified TM was added to [64Cu]CuCl2 (200 MBq, in 0.01 M HCl) and incubated for 30 min at 37 °C. The pH was adjusted to 5–6. Radiochemical yield and radiochemical purity were analyzed by radio-TLC (solid phase: iTLC-SG (Agilent), mobile phase: PBS) and radio-HPLC using a sample of the radiolabeled TMs in 2 mM aq. EDTA solution. Radiolabeled TMs were purified by spin filtration using Millipore AmiconUltra-4 (as described before) and PBS containing 2 mM EDTA and 0.0067% Dodecyl-β-D-maltoside (DDM). After that, the TMs were once again analyzed by HPLC.
The production of 64Cu was performed via proton irradiation of enriched 64Ni at a TR‐Flex cyclotron from Advanced Cyclotron Systems Inc (ACSI, Canada) and module‐assisted separation as described in detail recently [39 ].
The production of 64Cu was performed via proton irradiation of enriched 64Ni at a TR‐Flex cyclotron from Advanced Cyclotron Systems Inc (ACSI, Canada) and module‐assisted separation as described in detail recently [39 ].
3
Preparation of Isotope-Labeled SLII Constructs
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All SLII constructs used in this study were prepared by in vitro transcription using recombinant T7 polymerase that was overexpressed and purified from BL21 (DE3) cells. Synthetic DNA templates corresponding to the EV-A71 2231 and EV-D68 isolates, or mutant constructs were purchased from Integrated DNA Technologies (Coralville, IA). Transcription reactions were performed using standard procedures and consisted of 3 to 6 ml of reaction volumes containing unlabeled ribonucleotide triphosphates (rNTPs) or (C13/N15)–labeled rNTPs. Following synthesis, samples were purified to homogeneity by denaturing polyacrylamide gel electrophoresis (PAGE), excised from the gel, electroeluted, and desalted via exhaustive washing of the samples with ribonuclease-free water using a Millipore Amicon Ultra-4 centrifugal device. Samples were annealed by heating at 95°C for 2 min and flash-cooled on ice. Samples were subsequently concentrated and exchanged into 10 mM K2HPO4 (pH 6.5) and 20 mM KCl, 4 mM tris(2-carboxyethyl)phosphine (TCEP), and 0.5 mM EDTA using a Millipore Amicon Ultra-4 centrifugal filter device. The concentration of the samples was determined using the respective RNA theoretical molar extinction coefficient, and NMR samples ranged from 0.1 to 0.2 mM at 200 μl.
4
Protein Fractionation Using Amicon Filtration
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E64 was added to 50 kDa cutoff device (Millipore's Amicon® Ultra-4). The filtrated liquid was collected following centrifugation at 4,000 g (40 min). The procedure was repeated by adding deionized water until the filtrated liquid was colorless. The intercepted liquid was collected as the >50 kDa fraction, and the filtrated liquid was added to 10 kDa cutoff device (Millipore's Amicon® Ultra-4) for repeated centrifugation separation. The resultant filtrated liquid and intercepted liquid were collected as the <10 kDa fraction and the 10–50 kDa fraction, respectively. Each fraction was freeze-dried and stored at −80 °C.
5
Affinity Purification of Anti-BCNT-C Antibody
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GS(332–346) peptide, acetylated-EKKGYFEDRRPSANC-COOH (0.75 mg, 91.2% purity, obtained from AnyGen), was coupled to 1 mL of SulfoLink Coupling Resin (Thermo Fisher Scientific) packed in a column (MoBiTec) according to a manufacture’s protocol. The anti-BCNT-C Ab of 4.45 mg diluted in 10 mL with Binding/Wash buffer (20 mM PBS (pH 7.0) containing 0.05% sodium azide) passed through the column 5 times and a flow-through fraction was obtained. After washing the resin with 50 mL of Binding/Wash buffer, the bound fraction (GYFE fraction) was eluted with 6 mL of 100 mM Glycine (pH 3.0) and immediately neutralized with Neutralization buffer (1 M Tris-HCl (pH 8.5), 1.5 M NaCl, 5 mM EDTA). The protein concentrations of the binding fraction (GYFE fraction) and the flow-through fraction were determined by Bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific) using bovine serum albumin as a standard after buffer exchange and concentration by ultrafiltration using Amicon Ultra-4, 30 kDa (Merck Millipore). Their protein concentrations were 0.78 mg and 2.80 mg, respectively, with total recovery of 80.7%.
6
Purification of Bacteriophage fRuSau02
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The fRuSau02 lysate (5 × 1010 plaque-forming units (PFU)/mL) was ultrafiltrated with Amicon Ultra-4 (100 kDa) Centrifugal Filter Units (Merck Millipore, Billerica, MA, USA) to one quarter of the initial volume. Three volumes of chromatography buffer A (20 mM Tris-Cl, pH 7.5) were added and the ultrafiltration was repeated. The volume was adjusted with buffer A. The ultrafiltrated phage sample was then purified with ion exchange chromatography (IEX) using Äkta Purifier (GE Healthcare, Chicago, IL, USA) and a CIM QA-1 tube monolithic column with a 6-µm pore size (BIA Separations, Ajdovščina, Slovenia). The sample was injected to the column in buffer A, washed with buffer A containing 350 mM NaCl and eluted with buffer A with 450 mM NaCl. The phage-containing fractions of two purification batches were pooled, and an Amicon Ultra was used to concentrate the product and to change the buffer to TM (50 mM Tris, pH 7.5–10 mM Mg2SO4). Purified phage samples were stored at 4 °C.
7
Gelatin Zymography Assay for MMP Activity
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Conditioned media from cells grown in serum-free media for 72 h were collected and centrifuged for 10 min at 1500 rpm and 4 °C. Supernatants were concentrated using Amicon Ultra-4 centrifugal filters (Merck Millipore, Burlington, MA, USA), and protein content was evaluated with the standard Bradford procedure [55 (link)]. Samples were prepared using 3 µg of protein and non-reducing sample buffer, while gels for electrophoresis contained 0.1% gelatin. After electrophoresis, gels were washed with buffer containing Triton X-100 to remove the SDS and then incubated in detergent-free buffer for 12 h at 37 °C. Gels were then stained with Coomassie Brilliant Blue R for 30 min, followed by destaining to visualize areas of digestion seen as transparent spots on a blue background. Images of gels were captured with ChemiDoc (Bio-Rad, Hercules, CA, USA) and, after inversion of colors, the densitometry was performed using ImageLab software (ver. 6.0, Bio-Rad, Hercules, CA, USA). Obtained results were calculated as a fold change of MMP activity between the parental and resistant lines.
8
Bone Protein Extraction and Quantification
9
SARS-CoV-2 Spike Protein Expression
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pcDNA3.1 SARS-CoV-2 S D614G was a gift from Jeremy Luban (Addgene plasmid # 158075; http://n2t.net/addgene:158075 ; RRID: Addgene_158075). SARS-CoV-2 S D614G protein, RBDwt, RBDδ, and RBDο were produced in Expi293 cells (Thermo Fisher Scientific) and were purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously (24 (link), 25 (link)).
Briefly, Expi293 cells were cultured at 37 °C with 5% CO2 for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBDwt, RBDδ, or RBDο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.
Briefly, Expi293 cells were cultured at 37 °C with 5% CO2 for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBDwt, RBDδ, or RBDο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.
10
Quantifying Lipid-encapsulated Drug Delivery
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The STB-ME samples were placed in an ultrafiltration tube with an MW cut off of 10 kDa (Amicon® Ultra-4, Merck Millipore Ltd. Ireland) and centrifuged (4,000 rpm, 4°C) by a low-temperature centrifuge for 10 min. The filtrate was obtained from the bottom of the ultrafiltration tube by centrifugation, while the STB content of STB-ME was analyzed without centrifugation. The filtrates and the STB-ME samples were diluted with methanol in different proportions, and then the STB content was analyzed by HPLC (Section 2.3 ). The drug loading (DL) and encapsulation efficiency (EE) were calculated by the following two equations (Vieira et al., 2020 (link); Ma et al., 2022 (link)):
where is the mass of total STB in STB-ME, is the mass of free STB after centrifugation, and is the mass of STB and lipid.
where is the mass of total STB in STB-ME, is the mass of free STB after centrifugation, and is the mass of STB and lipid.
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