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Pd minitrap g 25

Manufactured by Cytiva

The PD MiniTrap G-25 is a pre-packed size exclusion chromatography column designed for desalting and buffer exchange of small sample volumes. The column comes pre-packed with Sephadex G-25 resin and is available in two sizes, 1 mL and 5 mL, to accommodate different sample volumes.

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6 protocols using pd minitrap g 25

1

VNAR Antibody Expression and Purification

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VNAR cDNAs prepared by gene synthesis were fused with human Fc or AGIA-His tandem tag and subcloned into the pcDNA3.4 expression vector using Gibson Assembly. VNAR antibodies were expressed using the Expi293F Expression System (Thermo Fisher Scientific, A14635), according to the manufacturer’s instructions. VNAR-Fc antibodies were purified using protein G Sepharose 4 Fast Flow (Cytiva), and then the buffer was exchanged with PBS using PD MiniTrap G-25 (Cytiva). VNAR-AGIA-His antibodies were purified via Ni Sepharose High Performance, and then buffer was exchanged with PBS via PD-10 column. Antibody concentration was determined using the extinction coefficient method (Gasteiger et al., 2005 (link)) with a NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified antibodies were frozen and stored at −30°C.
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2

Measuring IscS-IscU Binding Affinity

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To evaluate the Kd of the IscSU complex by native mass spectrometry, an aliquot of purified protein (as isolated IscU or IscS) was first exchanged into 250 mM ammonium acetate pH 8.0 using desalting columns (PD MiniTrap G25, Cytiva). The protein concentration was determined by absorbance and working solutions (200 μL) were prepared by combining aliquots of IscS and IscU to achieve the desired concentration or IscS/IscU ratio, then infused directly into the source of a Waters Synapt XS (Waters Inc.) mass spectrometer operating in the positive ion mode. Data were acquired and processed with Mass Lynx v4.2 (Waters Inc.) over the m/z range of 3000 to 8000 m/z with parameters as follows: dry gas flow 4 L min−1, nebuliser gas pressure 6 Bar, dry gas 150 °C, source temperature 80 °C, capillary voltage 3100 V, offset 30 V, cone voltage (isCID) 150 V, trap collisional energy 10 eV, and transfer collision energy 2 eV. Spectra were averaged, and the neutral mass spectrum calculated (90–125 kDa) using the MaxEnt1 deconvolution algorithm of MassLynx v4.2 (Waters Inc.). At least two datasets were acquired, expressed as fractional saturation, and then averaged prior to fitting with Dynafit (Biokin), as previously described.22 (link) The mass spectrometer was calibrated with sodium iodide. Average resolution for the measured range was 20 000 FWHM.
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3

Antibody-Drug Conjugate Formation via Cysteine Engineering

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10 μM h38C2_K99C, h38C2_K99Y and parental DVD-Fab targeting HER2 antigen were incubated with 50 μM compound 1, 2, 3 in PBS (pH 8.5) for 1 h at RT. Subsequently, 7.5 μg of the conjugation mixtures was loaded onto a 10-well NuPAGE 4–12% Bis-Tris Protein Gel. Fluorescent bands were first visualized by blue light on an E-gel Imager (Thermo Fisher) before the gel was stained with PageBlue Protein Staining Solution. To generate ADCs in DVD-IgG1 format, 10 μM h38C2_K99C DVD-Fab and h38C2_K99C DVD-IgG1 were incubated with 50 and 100 μM of compound 4 in PBS (pH 8.5) for 1 h at RT. For comparison, 10 μM h38C2 DVD-IgG113 (link) was incubated with 100 μM β-lactam-hapten-MMAF at RT for 4 h. Following incubation, free compounds were removed with a PD MiniTrap G-25 (Cytiva) column. Finally, the ADCs were concentrated with Amicon Ultra 0.5-mL Centrifugal Filters to >1 mg/mL in PBS (pH 7.4).
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4

Kinesin Protein Labeling and Storage

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Kinesin was labeled with ATTO 647N maleimide (AD 647N-41, ATTO-TEC) or Cyanine3B (Cy3B) maleimide (19380, Lumiprobe) overnight at 4 °C. Excess dye was removed from the reaction mixture by size-exclusion chromatography (PD MiniTrap G-25, 28-9180-07, Cytiva) according to the manufacturer’s protocol. The degree of labeling was determined by ultraviolet–visible spectroscopy (DS-11+ Spectrophotometer, DeNovix) and mass spectrometry (ESI, maXis II ETD, Bruker). Sucrose was added to the labeled protein in a concentration of 10% (w/v), and aliquots were flash-frozen in liquid nitrogen and stored at −80 °C.
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5

Preparation and Characterization of Fluorescent Liposomes

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Non-fluorescent liposomes were prepared as described above. 5(6)-Carboxyfluorescein (CF) (Merck; 100 mM CF stock, dissolved in 300 mM NaOH) was mixed in a 1:1 ratio with a buffer, containing 300 mM NaCl, and 100 mM Na2HPO4-NaH2PO4, pH 6. The dried lipid film was rehydrated with the 50 mM CF-stock solution, followed by sonication and extrusion. Finally, to remove the excess CF, the liposomes were applied onto a gravity flow desalting column (PD MiniTrap G25, Cytiva), pre-equilibrated with an elution buffer containing 150 mM NaCl, and 50 mM Na2HPO4-NaH2PO4, pH 6. Liposome content mixing fluorometry measurements were performed using a Varioskan LUX Fluorometer plate reader (ThermoFisher Scientific), similar to the R18-based assay in a 96-well format (200 µL reaction volume). Briefly, 490/515 nm excitation/emission wavelengths and 5 nm slits were used. The liposome mixture consisted of a 1:2 ratio between CF-encapsulating and empty LUVs (250 µM total lipid concentration), respectively, and measurements were performed in the elution buffer.
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6

Liposome Scrambling and Leakage Assays

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For the scrambling assay43 (link), NBD-labeled liposomes were prepared by adding 1% (w/v) nitrobenzoxadiazole (NBD)-labeled lipid (NBD-PE) to the lipid mixture. The scrambling assay was performed in a 100 μl quartz cuvette at room temperature (250 µM final lipid concentration). The NBD-labeled liposomes were incubated with CLIC5 (25 μM), and the NBD fluorescence was monitored before and following the addition of dithionite (to 5 mM) using 460/538 nm excitation/emission, respectively (Jasco FP-8500 spectrofluorometer). Additional dithionite (5 mM) and Triton X-100 (0.5% w/v) were added to dissolve the liposomes and allow complete quenching of the NBD fluorescence.
For the NBD-glucose leakage control assay43 (link), the lipid film was equilibrated with NBD-glucose-containing buffer (12.6 µM). Excess NBD-glucose was removed by passing the liposomes through a desalting column (PD MiniTrap G25, Cytiva) pre-washed with NBD-glucose-free buffer. Next, the experimental procedure was performed similarly to the scrambling assay, with first dithionite addition (to 1 mM), following second dithionite addition and (1 mM) and Triton X-100 (0.5% w/v) to quench the NBD-glucose signal completely.
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