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Sephadex g 25 resin

Manufactured by Cytiva
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Sephadex G-25 is a dextran-based size exclusion chromatography resin. It is designed to separate molecules based on their size and molecular weight, allowing for the separation and purification of proteins, peptides, and other biomolecules from smaller molecules, salts, and other contaminants.

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Lab products found in correlation

Cocl2 6h2o, by Merck Group (1 mentions) Dithiothreitol (dtt), by GoldBio (1 mentions) Tris hcl, by Avantor (1 mentions) Tris 2 carboxyethyl phosphine tcep, by GoldBio (1 mentions) Guanidine hcl, by Avantor (1 mentions) Glutamine gln, by Thermo Fisher Scientific (1 mentions) Pet his6 sumo tev lic cloning vector, by Addgene (1 mentions) Deoxycytidine, by Merck Group (1 mentions) Ampicillin trisodium salt, by Merck Group (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions) Luria broth, by Merck Group (1 mentions) Q sepharose, by GE Healthcare (1 mentions) Ni nta agarose resin, by Qiagen (1 mentions) E coli bl21 de3 competent cells, by New England Biolabs (1 mentions) Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions) Mgso4, by Merck Group (1 mentions) Adenosine triphosphate (atp), by Merck Group (1 mentions) Pd 10 desalting column, by Cytiva (1 mentions) Panitumumab, by LI COR (1 mentions) Ir700 nhs ester, by LI COR (1 mentions)

7 protocols using sephadex g 25 resin

1

Labeling of PHD2 Protein with Alexa Fluor 647

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A 500 µl aliquot of 1 mg/ml PHD2 in Labeling Buffer was labeled with Alexa Fluor 647 N-hydroxysuccinimide ester dye (Thermo Scientific) by addition of a 4:1 molar ratio of dye:PHD2. Labeling was conducted at room temperature for 1 h in the dark. Excess dye was then removed first by the passage of the sample through a PD-10 desalting column containing Sephadex G-25 resin (Cytiva) equilibrated with Dialysis Buffer and then any remaining excess dye was removed via repeated 10-fold dilution in Dialysis Buffer and subsequent concentration in an Amicon 10 kDa cut off centrifugal filter until the dye could no longer be detected in the concentrator flow through by Absorbance at 650 nm. This procedure should result in Alexa 647 labeled PHD2 (PHD2-647) with a labeling efficiency of 1-2 dye molecules per protein molecule. PHD2-647 was diluted to a concentration of 10 µM in Dialysis Buffer, aliquots were used immediately, or flash frozen in liquid N2 and stored at −80 °C until needed.
Ferens F.G., Taber C.C., Stuart S., Hubert M., Tarade D., Lee J.E, & Ohh M. (2024). Deficiency in PHD2-mediated hydroxylation of HIF2α underlies Pacak-Zhuang syndrome. Communications Biology, 7, 240.
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2

Conjugation of Peptides to CPMV Nanoparticles

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CBP-biotin (N-GWRVSEFGGGSK/biotin/-C) and CBP-FITC (N-GWRVSEFGGGSK/FITC/-C) were synthesized by GenScript with 75% purity. Binding of CBP-biotin or CBP-FITC peptide to CPMV was performed by incubating a 500-fold molar excess of peptide per CPMV; the mixture was allowed to react overnight at 4 °C. Complexed product, i.e., CPMV-CBP-biotin and CPMV-CBP-FITC, was purified by using PD MiniTrap desalting columns with Sephadex G-25 resin (Cytiva) followed by 100 kDa Amicon Ultra-0.5 centrifugal unit (dual-step purification). The concentration of the final product was determined by Nanodrop as described above. Particle integrity was validated by DLS and TEM as described above.
Chan S.K, & Steinmetz N.F. (2021). Isolation of Cowpea Mosaic Virus-Binding Peptides. Biomacromolecules, 22(8), 3613-3623.
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3

Synthesis of Protein-Coated Iron Oxide Nanoparticles

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Prot-IONPs were synthesized through a coprecipitation approach
based on a previously reported method.52 (link) Briefly, 1 mL of the protein scaffold at 45 μM was mixed with
a 2:1 mixture of FeCl2·4H2O and FeCl3·6H2O (6 eq. respect to Glu metal-binding
residues) under a nitrogen atmosphere. Then, the reaction mixture
was incubated for 30 min at room temperature and a 0.1 mM NaOH solution
was added until a pH value equal to 10 was reached. Then the reaction
was sonicated for 10 min at 65 °C, and a solution of sodium ascorbate
(10 equiv with respect to iron ions) was added to the reaction mixture,
which was again sonicated for 10 min at 65 °C. Finally, Prot-IONPs
were purified by gel filtration using a PD-10 column containing Sephadex
G-25 resin (Cytiva).
Aires A., Fernández-Afonso Y., Guedes G., Guisasola E., Gutiérrez L, & Cortajarena A.L. (2022). Engineered Protein-Driven Synthesis of Tunable Iron Oxide Nanoparticles as T1 and T2 Magnetic Resonance Imaging Contrast Agents. Chemistry of Materials, 34(24), 10832-10841.
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4

Synthesis and Purification of Proteins

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Co(Cl)2·6H2O, CH3I, and iPrBr were obtained from Sigma-Aldrich; Co(OAc)2·3H2O was obtained from Mallinckrodt; 4-methylpyridine (pyMe) and 4-vinylpyridine (pyv) were obtained from Oakwood Chemicals; and all deuterated solvents were purchased from Cambridge Isotope Laboratory. Dry solvents were obtained from a Jorg. C. Meyer solvent purification system. Ni-NTA resin was obtained from Cube BioTech and Thermo Fisher Scientific, while Sephadex G25 resin was obtained from Cytiva. Guanidine HCl and Tris HCl were purchased from VWR. Dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Gold-Bio. Isopropylthio-β-D-galactoside (IPTG), 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), and glutamine (Gln) were purchased from Thermo Fisher Scientific. All chemicals were used as ordered without further purification. All buffers were prepared using 18.2 MΩ cm resistivity water from a Milli-Q water purification system. The pET His6 SUMO TEV LIC cloning vector was purchased from Addgene, and E. coli BL21 (DE3) cells were purchased from Novagen. TEV protease was expressed and purified according to literature protocols.
Neuwirth C., Madec S., Siebor E., Pechinot A., Duez J.M., Pruneaux M., Fouchereau-Peron M., Kazmierczak A, & Labia R. (2001). TEM-89 β-Lactamase Produced by a Proteus mirabilis Clinical Isolate: New Complex Mutant (CMT 3) with Mutations in both TEM-59 (IRT-17) and TEM-3. Antimicrobial Agents and Chemotherapy, 45(12), 3591-3594.
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5

Fluorinated Tyrosine Incorporation Assay

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Luria broth, ampicillin trisodium salt, phenylmethylsulfonyl fluoride (PMSF), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), MgSO4, adenosine triphosphate (ATP), deoxycytidine, cytidine diphosphate (CDP) and alkaline phosphatase (AP, calf intestine) were purchased from Sigma-Aldrich. Isopropyl β-D-1-thiogalactopyranoside (IPTG), L-arabinose, kanamycin (Km), chloramphenicol (Cm), dithiothreitol (DTT) were purchased from GoldBio. DEAE and Q-sepharose resins were obtained from GE Healthcare Life Sciences. Ni-NTA agarose resin was purchased from Qiagen. PD-10 desalting columns packed with Sephadex G-25 resin (10 mL) were obtained from Cytiva. The primers for site-directed mutagenesis were obtained from Integrative DNA Technologies (IDT). E. coli BL21(DE3) competent cells were obtained from New England Biolabs (NEB). [5-3H]-CDP was purchased from ViTrax. Emulsifier-Safe scintillation cocktail was obtained from Perkin-Elmer. 2,3,5-Trifluorotyrosine (2,3,5-F3Yxxx, where xxx indicates the residue number) and 3,5-difluorotyrosine (3,5-F2Yxxx, where xxx indicates the residue number), thioredoxin (TR) and thioredoxin reductase (TRR) were available from a prior study. Tricarbonyl(1,10-phenanthroline)(4-bromomethylpyridine) rhenium(I) hexafluorophosphate ([Re]-Br) was synthesized via the previously modified procedure.22 (link)
Cui C., Song D.Y., Drennan C.L., Stubbe J, & Nocera D.G. (2023). Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase. Journal of the American Chemical Society, 145(9), 5145-5154.
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6

Fluorescent Antibody Conjugates for Imaging

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Panitumumab, anti-human Epidermal Growth Factor Receptor (hEGFR) antibody, was obtained from Amgen (Thousand Oaks, CA, USA; RRID AB_2459650). Anti-mouse CD29 antibody (clone IM7; RRID AB_2687714) was purchased from Bio X Cell (West Lebanon, NH, USA). Panitumumab (1 mg, 6·7 nmol) or anti-mouse CD29 antibody (1 mg, 6·7 nmol) was incubated with five-fold molar excess of IR700 NHS ester (10 mM in DMSO; LI-COR Biosciences, Lincoln, NE, USA) in 100 mM Na2HPO4 solution (pH 8·5) for 1 h at room temperature. The mixture was purified with PD-10 columns containing Sephadex G25 resin (Cytiva, Marlborough, MA, USA). The resulting AbPCs were abbreviated as pan-IR700 or CD29-IR700, respectively. 3′-OMe-DiR was synthesised as described previously.16 (link)
Fukushima H., Furusawa A., Takao S., Matikonda S.S., Kano M., Okuyama S., Yamamoto H., Choyke P.L., Schnermann M.J, & Kobayashi H. (2024). Phototruncation cell tracking with near-infrared photoimmunotherapy using heptamethine cyanine dye to visualise migratory dynamics of immune cells. eBioMedicine, 102, 105050.
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7

Antibody Labeling and Purification

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We labeled the affinity-purified antibodies, at 250 μg/reaction, with the donor (europium [Eu]) and acceptor (Alexa Fluor 647 [AF647]) using a QuickAllAssay Eu-chelated protein labeling kit (BN Products and Services Oy) and Alexa Fluor 647 NHS (N-hydroxysuccinimide) ester (Thermo Scientific) according to the manufacturer’s instructions. A disposable PD-10 desalting column with Sephadex G-25 resin (Cytiva) served to remove nonreacted fluorophores, and an Amicon Ultra 0.5-ml 50-kDa-NMWL centrifugal filter (Millipore/Merck) was used for concentrating the labeled antibodies, which were then stored aliquoted at −80°C until use.
Rusanen J., Kareinen L., Szirovicza L., Uğurlu H., Levanov L., Jääskeläinen A., Ahava M., Kurkela S., Saksela K., Hedman K., Vapalahti O, & Hepojoki J. (2021). A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection—SARS-CoV-2 as a Proof of Concept. mBio, 12(3), e00902-21.
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