Pd minitrap g 25 desalting column

Manufactured by GE Healthcare

Sourced in United States

The PD MiniTrap G-25 is a desalting column designed for the rapid and efficient removal of salts, buffers, and other low molecular weight compounds from protein samples. The column utilizes Sephadex G-25 resin to separate the components based on their molecular size, allowing the target protein to be collected in a small volume while the unwanted substances are retained.

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Lab products found in correlation

Nhs peg4 sat, by Thermo Fisher Scientific (2 mentions) Maleimide peg4 nhs, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Nhs peg4 mal, by Thermo Fisher Scientific (1 mentions) Tla 55 rotor, by Beckman Coulter (1 mentions) Nexion 350d, by PerkinElmer (1 mentions) Irdye 800cw, by LI COR (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Shuffle t7, by New England Biolabs (1 mentions)

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G-25 column (60 citations) Desalting column (49 citations) PD MiniTrap G-25 (35 citations)

9 protocols using pd minitrap g 25 desalting column

Reconstitution of clMagR Protein

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The purified wild-type clMagR and mutant proteins (400 μmol/L) were incubated with 10 mmol/L Na
₂S
₂O
₄ and 10 mmol/L ethylenediaminetetraacetic acid (EDTA) overnight at 4 °C in TBS buffer (20 mmol/L Tris, 150 mmol/L NaCl, pH 8.0) to remove iron-sulfur clusters. The mixture was desalted using a PD MiniTrap G-25 desalting column (GE Healthcare, USA). The obtained protein samples were labeled as “apo-clMagR”. Apo-clMagR was incubated with 5 mmol/L dithiothreitol (DTT) for 30 min at 4 °C, then with Fe(NH
₄)
₂(SO
₄)
₂ (1.6 mmol/L) and Na
₂S (1.6 mmol/L) overnight at 4 °C. The mixture was desalted using a PD MiniTrap G-25 desalting column (GE Healthcare, USA) and the obtained protein samples were labeled as “chem re clMagR”.

Zhou Y., Tong T., Wei M., Zhang P., Fei F., Zhou X., Guo Z., Zhang J., Xu H., Zhang L., Wang S., Wang J., Cai T., Zhang X, & Xie C. (2023). Towards magnetism in pigeon MagR: Iron- and iron-sulfur binding work indispensably and synergistically. Zoological Research, 44(1), 142-152.

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Purification and Characterization of Methionine Sulfoxide Reductase

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Isopropyl β-d-thiogalactopyranoside (IPTG), DNase I, phenylmethylsulfonyl fluoride (PMSF), protease inhibitor EDTA-free mixture, DTT, bovine pancreas insulin, DTNB (Ellman's reagent), β-NADPH–reduced tetra(cyclohexylammonium) salt (NADPH), methionine sulfoxide (MetO), antibiotics, and all the chemical reagents used for buffer preparation were from Sigma-Aldrich Chemie GmbH. PD MiniTrap G-25 desalting columns and all other columns used for chromatography were from GE Healthcare.

Napolitano S., Reber R.J., Rubini M, & Glockshuber R. (2019). Functional analyses of ancestral thioredoxins provide insights into their evolutionary history. The Journal of Biological Chemistry, 294(38), 14105-14118.

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Conjugation of Serum Albumin to TMV

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Human serum albumin (SA; Sigma Aldrich) was conjugated to the solvent-exposed exterior surface of TMV-lys through a PEG linker. SA was first conjugated using NHS-PEG₄-SAT (ThermoFisher) at a 1-to-1 ratio in 10 mM potassium phosphate buffer, pH 7.4 containing 10% v/v DMSO overnight at RT. De-acetylation solution (0.5 M hydroxylamine, 25 mM EDTA in PBS, pH 7.2–7.5) was added to the reaction at a final concentration of 10% v/v to de-protect the thiol group and this de-protection reaction was carried for 2 hours at RT. TMV-lys was conjugated using maleimide-PEG₄-NHS (ThermoFisher) at 10 equivalents PEG per TMV-lys coat protein in 10 mM potassium phosphate buffer, pH 7.4 containing 10% v/v DMSO for 2 hours at RT. The resulting SA-PEG₄-SH conjugates and MAL-PEG₄-TMV conjugates were purified through a PD MiniTrap G-25 desalting columns (GE). Purified SA-PEG₄-SH was then reacted with purified MAL-PEG₄-TMV at a ratio of 6 equivalents of SA-PEG₄-SH per TMV coat protein overnight at room temperature. The reaction was quenched for 1 hour at RT by addition of excess glycine and l-cysteine. SA-PEG₈-TMV (Fig. 1A) was then purified by ultracentrifugation at 55,000 rpm for 3 hours on a 40% w/v sucrose cushion. Conjugation was verified and quantified using SDS-PAGE analysis.

Gulati N.M., Pitek A.S., Steinmetz N.F, & Stewart P.L. (2017). Cryo-Electron Tomography Investigation of Serum Albumin-Camouflaged Tobacco Mosaic Virus Nanoparticles. Nanoscale, 9(10), 3408-3415.

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TMV-PEG4 Particle Synthesis

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TMV-PEG₄ particles were obtained by conjugation of NHS-PEG₄-mal (22114; Thermo Fisher) to the external surface of TMV-Lys. The reaction was performed by mixing TMV-Lys (2 mg/mL final concentration) and NHS-PEG₄-mal at 10 equiv per TMV-Lys coat protein in 0.01 M potassium phosphate buffer pH 7.4 containing 10% (v/v) DMSO; the reaction was allowed to proceed overnight at RT. Then the reaction was quenched by addition of excess glycine/L-cysteine, and quenching was allowed to proceed for 1 h at RT. The products were then purified using PD MiniTrap G-25 desalting columns (28-9180-08; GE).

Pitek A.S., Wang Y., Gulati S., Gao H., Stewart P.L., Simon D.I, & Steinmetz N.F. (2017). Elongated Plant Virus-Based Nanoparticles for Enhanced Delivery of Thrombolytic Therapies. Molecular pharmaceutics, 14(11), 3815-3823.

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Streptavidin-Conjugated Tobacco Mosaic Virus

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Human or mouse SA (Sigma-Aldrich or Bioworld, respectively) was conjugated to the surface of TMV targeting Lys side chains. First, SA was conjugated with NHS-PEG₄-SAT (Thermo Fisher) at 1:1 ratio in phosphate buffered saline (PBS; 0.01 M Na₂HPO₄, 0.0018 M KH₂PO₄, 0.0027 M KCl and 0.137 M NaCl, pH 7.4) containing 10% (v/v) DMSO overnight at RT. To de-protect the thiol group, 0.5 M hydroxylamine, 25 mM EDTA in PBS, pH 7.2–7.5, was added at a final concentration of 10% (v/v) and incubated for 3 h at RT. Separately, TMV was conjugated with maleimide-PEG₄-NHS (Thermo Fisher) at 10 equiv of PEG per coat protein in 10 mM potassium phosphate buffer, pH 7.4, containing 10% (v/v) DMSO, for 3 h at RT. The resulting maleimide-PEG₄-TMV conjugates were purified twice through PD MiniTrap G-25 desalting columns (GE) and combined with previously prepared SA-PEG₄-SH at 6 equiv per CP and reacted overnight at RT to yield SA-TMV particles. The reaction was quenched by addition of excess glycine and l-cysteine for 1 h at RT. The resulting constructs were then purified by ultracentrifugation at 112 000g (50 000 rpm using TLA-55 rotor, Beckman Coulter) for 1 h on a 40% (w/v) sucrose cushion in PBS. The final samples were resuspended in PBS, pH 7.4, to yield a concentration of 10 mg/mL of SA-TMV; aggregates were removed by centrifugation at 7500g for 5 min.

Pitek A.S., Hu H., Shukla S, & Steinmetz N.F. (2018). Cancer Theranostic Applications of Albumin-Coated Tobacco Mosaic Virus Nanoparticles. ACS applied materials & interfaces, 10(46), 39468-39477.

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Determining Zinc-Binding Stoichiometry of ZnT8 Variants

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Concentrated apo‐protein samples of both variants were prepared and diluted with ≥ 18.2 MΩ.cm water (ELGA LabWater, High Wycombe, UK). These samples were analysed for zinc and nickel with ICP‐MS (Perkin Elmer Life Science, Waltham, MA, USA, model NexION 350D) and were shown to have reduced metal content compared with protein samples that had not been incubated with additional TCEP and EDTA.
To measure zinc‐binding stoichiometry, ZnT8cR and ZnT8cW samples were prepared in 10 mm Tris/HCl, pH 8, 60 mm NaCl and 20 mm sucrose. Samples of each isoform were diluted to 10 μm. Each protein sample was then incubated with 0, 1, 2, 4 or 10 molar equivalents of Zn²⁺ (ZnCl₂) for 10 min at 21 °C, followed by gravity‐flow gel filtration using PD MiniTrap G‐25 desalting columns (GE Healthcare) to remove excess zinc. Samples were then diluted with ≥ 18.2 MΩ.cm water and analysed for zinc and nickel using ICP‐MS.

Parsons D.S., Hogstrand C, & Maret W. (2018). The C‐terminal cytosolic domain of the human zinc transporter ZnT8 and its diabetes risk variant. The Febs Journal, 285(7), 1237-1250.

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Agrobacterium-Mediated Transient Expression

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The entire coding sequences with or without three FLAG tags (DYKDHDGDYKDHDIDYKDDDDK) at the C-terminus were cloned into the multiple cloning sites of the binary vector pEl2Ω [28 (link)]. The constructs were used to transform Agrobacterium tumefaciens strain GV3101 by electroporation. Transformed cells were cultured at 30°C to mid-log phase and collected by centrifugation. After washing with 10 mM MES-NaOH (pH 5.6), 10 mM MgCl₂, and 150 μM acetosyringone, the cells were suspended with the same solution to A₆₀₀ = 0.1. After a 2-h incubation at room temperature, the cells were infiltrated into the leaves of N. benthamiana using a syringe without a needle.
For the ICS activity assays in vitro, leaves infiltrated with Agrobacterium were collected at 2 days after infiltration and ground in buffer A containing cOmplete, EDTA-free protease inhibitor cocktail (Roche). After centrifugation, the supernatants were desalted using a PD MiniTrap G-25 desalting column (GE Healthcare) equilibrated with buffer A in accordance with the manufacturer’s recommendations, and used as a crude protein fraction.
For ICS activity assays in planta, Agrobacterium cells carrying each ICS gene were mixed with different volumes of Agrobacterium cells carrying the TP^SS-IPL^PmsB, as indicated in Figure 4B. Two days later, the levels of total SA were determined as described previously [29 (link)].

Yokoo S., Inoue S., Suzuki N., Amakawa N., Matsui H., Nakagami H., Takahashi A., Arai R, & Katou S. (2018). Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties. Bioscience Reports, 38(2), BSR20171457.

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Quantifying Staphylococcus aureus IgG Binding

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S. aureus isolates were grown overnight in TSB, diluted 1:100 in fresh medium and cultured until the mid-exponential growth phase (OD₆₀₀ ∼0.5). Next, the cells were coated with complement by adding serum (end concentration 20%) and incubation was continued for 30 min. After this incubation, the bacteria were washed with phosphate-buffered saline (PBS). High-binding ELISA plates for fluorescence measurements (Greiner Bio-one) were coated with 5 × 10⁶ colony forming units (CFU) per well in PBS for 18 h at 4°C. Plates were blocked with 4% BSA in PBS with 0.05% Tween-20 (PBST). Surface-bound IgG Fc-binding proteins of S. aureus (i.e., Spa and Sbi) were saturated with 100 μg/mL normal rabbit immunoglobulin fraction (DAKO) in PBST containing 1% BSA. The humAb 6D4 was labeled with IRDye 800CW (LI-COR Biosciences, Bad Homburg, Germany) by incubation for 2 hours with 20 µg of IRDye 800CW per mg of protein in PBS (pH 8.5). The mix was desalted following the manufacturer's instructions with a PD minitrap G-25 desalting column (GE Healthcare, Germany). The resulting 6D4–800CW was stored in the dark at 4°C. To quantify the binding of 6D4–800CW to serum-incubated whole cells with added SCIN, the plates were incubated with 300 ng/mL 6D4–800CW in PBS for 30 min, washed thrice with PBS and scanned with the Odyssey infrared imaging system (Li-Cor Biosciences) for fluorescence at 800 nm.

Hoekstra H., Romero Pastrana F., Bonarius H.P., van Kessel K.P., Elsinga G.S., Kooi N., Groen H., van Dijl J.M, & Buist G. (2017). A human monoclonal antibody that specifically binds and inhibits the staphylococcal complement inhibitor protein SCIN. Virulence, 9(1), 70-82.

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Cloning and Expression of ICS Proteins

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The coding sequences without the putative chloroplast TP (Supplementary Figure S1) were amplified by PCR with NdeI at the 5′-end and XhoI or SalI at the 3′-end, and cloned into the corresponding sites of a pET28a vector (Novagen), allowing for the production of each ICS with a His₆ tag at the N-terminus. The resulting constructs were used to transform E. coli strain SHuffle T7 (New England Biolabs). Transformed cells were cultured at 30°C to mid-log phase, and the expression of recombinant proteins was induced by the addition of isopropyl β-d-thiogalactopyranoside at the concentrations indicated: 0.4 mM for AtICS1, OsICS, and their derivatives; 0.04 mM for NtICS; 0.01 mM for NbICS; and 0.5 mM for vector control and EntC. The cells were collected using a brief centrifugation after an 18-h culture at 20°C, resuspended in extraction buffer A (0.1 M Tris/HCl, pH 7.5, 1 mM DTT, 10% glycerol) containing cOmplete, EDTA-free protease inhibitor cocktail (Roche) and sonicated on ice. After centrifugation, the supernatants were desalted using a PD MiniTrap G-25 desalting column (GE Healthcare) equilibrated with buffer A in accordance with the manufacturer’s recommendations, and used as a crude protein fraction.

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