Amicon ultra 15 centrifugal filter unit

Shell proteins were extracted from 20 g of shells from about 60 individuals. First, shells were treated with sodium hypochlorite to remove any surface contaminants and then washed with ultrapure water to remove sodium hypochlorite. Second, washed shells were dissolved in an aqueous solution of 460 mL of 0.5 M ethylenediaminetetraacetic acid (EDTA), the pH adjusted to 8.0 using NaOH. After 48 h of incubation at 4 °C, the supernatant was decanted, and EDTA was removed with Amicon ultra-15 centrifugal filter units (Merck Millipore, Burlington, USA). This EDTA-soluble protein mixture is denominated the soluble fraction. The precipitate was dissolved in an aqueous solution containing 7 M urea, 2 M thiourea, 3% 3-[(3-cholamidopropyl)dimetilaminio] propanesulfonate (CHAPS), and 1% Triton X-100), and after 24 h of incubation at 4 °C, the preparation was centrifuged at 20,000 g, at 4 °C for 1 h. The supernatant was decanted, and urea and other salts were removed with Amicon ultra-15 centrifugal filter units (Merck Millipore, Burlington, USA). Th protein mixture solubilized by this procedure is referred to as the insoluble fraction. The amounts of soluble and insoluble fractions, in aqueous and CHAPS solutions, respectively, were quantified using a Qubit Fluorometer (Thermo Fisher Scientific, Massachusetts, USA).

Albumin was purified from mouse serum (Kohsin Bio Ltd., Saitama, Japan) using the modified Cohn method [4 (link)] and then eluted from the column using 25 mM acetate buffer (pH 4.5) and neutralized to pH 7.2. Albumin was concentrated in centrifugal filter units with a nominal molecular weight limit of 50 kDa (Amicon Ultra-15 Centrifugal Filter Units; Merck, Darmstadt, Germany). Albumin purification was confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined using the Bradford Ultra Total Protein Quantitation Kit (Abcam, Cambridge, UK) and adjusted to 5% (w/v) for administration. The albumin solution was stored at − 30 °C until further use.
HES powder (130/0.4/9; average molecular weight: 130 kDa; Fresenius Kabi, Bad Homburg, Germany) was used. HES labeled with FITC was prepared as previously described [4 (link)]. Low molecular weight (< 3 kDa) HES fractions were removed using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Units, Merck).

In this proof-of-concept study, we examined the role of the secreted SIFα and IFNα proteins in pancreatic cancer cells. For this, we expressed SIFα and IFNα in 293T cells. The supernatants (14 ml) containing the secreted interferons were collected and centrifuged at 4000 rpm for 10 min to get rid of cell debris. The supernatants were then passed through Amicon Ultra-15 centrifugal filter Unit (MW cut off 30 KD, EMD Millipore, CA) to get rid of large molecules. The flow-through containing the interferons (24.4 KD and 21.6 KD, respectively) was loaded onto Amicon Ultra-15 centrifugal filter Unit (MW cut off 10 KD, EMD Millipore, CA) to remove proteins with small molecule size. Interferons were measured with ELISA kit (PBL, CA). Based on the ELISA data, supernatants that contain the equal amount of secreted interferons were used for inhibition in cell growth.

Purification of PfCyRPA was carried out on an ÄKTA Explorer 100 System (Cytiva). Cell culture bulk was harvested, filtered through 0.45- and 0.22-μm Sartopore 2 Midicap Filter Cartridges 10 (Sartorius), and concentrated with a Sartocon disposable PES membrane 2 × 0.1 m², 10 kDa (Sartorius). The concentrated sample was filtered through a Nalgene cup of 0.2 μm (Thermo Scientific). PfCyRPA His₆- and His₄-tagged proteins were purified by immobilized metal ion affinity chromatography on a Histrap HP column (5 mL volume; Cytiva), while PfCyRPA C-tag tagged protein was purified on a CaptureSelect™ C-tagaffinity matrix chromatography column (5 mL volume; Thermo Fisher). Column eluates were concentrated using an AmiconUltra 15 Centrifugal Filter Unit 10 kDa (Merck Millipore), filtered through 0.2 μm, and applied to a HiLoad 26/60 Superdex 75 gel permeation column (GE Healthcare). The eluates were concentrated using an AmiconUltra 15 Centrifugal Filter Unit 10 kDa (Merck Millipore) and filtered through 0.2-μm filter. The final sample was stored in 50 mM Tris–HCl, pH 7.4 and 150 mM NaCl buffer, aliquoted, and stored at −80°C.

Both original and meditope-enabled anti-HER2^{1 (link)}, M5A⁶ , OKT3, and pertuzumab (Supplementary Methods) light and heavy chain genes were purchased from Atum in pJ609 and pJ607 expression vectors, respectively. They were sub-cloned into vector 253074 (Atum) for expression. Ile83Glu substitution on the light chain of the meditope-enabled anti-HER2 mAb to increase meditope-binding affinity was generated using standard site-directed mutagenesis and verified by DNA sequencing.
mAbs were produced using the ExpiCHO Expression System (Gibco). The vectors were co-transfected into ExpiCHO cells at 1 µg total plasmid DNA per 6 million cells. At 18 h post-transfection, ExpiFectamine, CHO Enhancer, and ExpiCHO Feed were added to the flask and then transferred to a 32 °C incubator (Max Titer Protocol). The culture supernatant was collected at 14 days post-transfection. The mAbs were purified from the culture supernatants using Protein G agarose beads (Genscript) and further purified on a Superdex HiLoad 16/60 75 or 200 pg column (GE Healthcare). The sized mAbs were concentrated and buffer-exchanged into PBS with Amicon Ultra-15 centrifugal filter units (Millipore) and stored at 4 °C.

The 293 T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10 % (vol/vol) fetal bovine serum and maintained at 37 °C at a concentration of 6,000,000 cells and transfected using Lipofectamine 2000 reagent (Invitrogen) with 3 μg GIPZ-shROR, 3 μg pMD2.D, and 6.0 μg PsPax. After incubation overnight with 293 T cells, the media was replaced with 5 mL fresh medium. The virus-containing supernatants were collected at 48 h and 72 h after transfection and then mixed and filtered through a 0.45 μm cellulose acetate filter (Sartorius). The viral supernatants were concentrated with Amicon Ultra-15 Centrifugal Filter Units (Millipore, USA) at 4 °C and spun at 5,000 rpm for 30 min. The colonies with GFP expression were selected for subsequent culture after incubation with 4 g/mL puromycin for 2 weeks.