BG505 SOSIP MD39 (250 μg) was incubated with 600 μg of Rh.33104 mAb.1 Fab and 600 μg of RM20A3 Fab (6 (link), 46 (link)) at room temperature overnight. The complex was SEC purified using a HiLoad 16/600 Superdex pg200 (GE Healthcare) column, with TBS as a running buffer. SEC fractions corresponding to the complex were pooled and concentrated to 6.0 mg/ml using an Amicon filter unit with 10-kDa cutoff (EMD Millipore).
Amicon filter unit
Manufactured by Merck Group
Sourced in United States, Germany
Amicon filter units are laboratory equipment designed for filtration and concentration of solutions. They utilize a semi-permeable membrane to separate components of a mixture based on their molecular size. The core function of these units is to isolate and concentrate specific molecules or particles from a sample.
Automatically generated - may contain errors
Lab products found in correlation
Sepharose 4b beads, by Merck Group (2 mentions)
Hitrap, by Cytiva (2 mentions)
Hiload 16 600 superdex pg200, by GE Healthcare (1 mentions)
Zeba column, by Thermo Fisher Scientific (1 mentions)
Axs 1114542 5, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Mab4067, by R&D Systems (1 mentions)
Pierce biotinylated protein interaction pull down kit, by Thermo Fisher Scientific (1 mentions)
Dynabeads m 280 tosylactivated, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Ni nta, by Qiagen (1 mentions)
Sp sepharose, by GE Healthcare (1 mentions)
Flag affinity chromatography, by Merck Group (1 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Hiload 26 600 superdex 75 column, by GE Healthcare (1 mentions)
21 protocols using amicon filter unit
1
Purification of BG505 SOSIP complex
2
Large-scale rAAV Production and Purification
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Recombinant AAV vectors were produced by calcium phosphate triple transfection of plasmids in HEK293 cells. For large-scale preparation, approximately 8.5 × 108 cells were transfected. rAAV was purified by two rounds of CsCl sedimentation followed by dialysis29 , which took a period of 7 days. For small-scale preparation, approximately 1.7 × 108 cells were transfected. rAAVs were purified using iodixanol gradient centrifugation30 (link). Briefly, HEK293 cells were detached by vigorously shaking the culture vessel. The cell suspension underwent three cycles of freezing (dry ice/ethanol bath) and thawing (37 °C water bath) for cell lysis and subsequent benzonase treatment. After centrifugation, the supernatant was transferred to an ultracentrifugation tube containing a discontinued gradient of 15, 25, 40, and 60% of iodixanol (Accurate Chemical, Cat. No. AN1114542). Gradient centrifugation was carried out at 504,000 × g for 70 min at 20 °C. The rAAV vectors at the 40–60% interface were collected and subjected to desalting using a Zeba column (Thermo Fisher Scientific, Cat. No. 89894) and concentrated using an Amicon Filter Unit (EMD Millipore, Cat. No. UFC910024). The entire procedure can be finished within 1 day. All rAAV vectors were titrated by droplet digital PCR (ddCPR, Bio-Rad) for genomes and silver staining of capsid proteins.
3
Production and Purification of Diverse AAV Vectors
4
Ultrafiltration-based Protein Separation
5
Efficient AAV Vector Production
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AAVs were prepared by the Boston Children’s Hospital Viral Core. AAV vectors were purified by discontinuous iodixanol gradients (Cosmo Bio; AXS-1114542-5) and concentrated with a Millipore Amicon filter unit (UFC910008, 100 kDa). AAV titers were determined by quantitative real-time PCR assays.
6
Evaluating Cell Proliferation, Motility, and Invasion
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EO771 mammary tumor cells (CH3 BioSystems, Amherst, NY, USA) [47 (link)] and astrocytes (Cell Biologics, Chicago, IL, USA) were grown in DMEM (Corning, Inc., Corning, NY, USA), MLO-A5 osteocyte-like cells (obtained from Dr. L. Bonewald at Indiana University, Indianapolis, IN, USA), and bone marrow-derived MSCs (harvested from C57BL/6 mice) were cultured in αMEM (Gibco, Carlsbad, CA, USA). The culture media were supplemented with 10% fetal bovine serum and antibiotics (50 units/mL penicillin, and 50 µg/mL streptomycin; Life Technologies, Carlsbad, CA, USA), and cells were incubated at 37 °C with 5% CO2. To test the effect of TGFβ, EO771 cells were treated with TGFβ (500 ng/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 1 day.
CM was prepared from MLO-A5 osteocytes and astrocytes at ~80% confluence after 24 h incubation and an Amicon filter unit with a cutoff mass at 3 kDa (Sigma-Aldrich, St. Louis, MO, USA) was used to remove microparticles and condense it by 10-fold. Cellular proliferation was examined using an MTT assay, and a wound-healing scratch assay and a Transwell invasion assay were conducted to evaluate cell motility and invasion capability, respectively, using the procedure previously described [48 (link)].
CM was prepared from MLO-A5 osteocytes and astrocytes at ~80% confluence after 24 h incubation and an Amicon filter unit with a cutoff mass at 3 kDa (Sigma-Aldrich, St. Louis, MO, USA) was used to remove microparticles and condense it by 10-fold. Cellular proliferation was examined using an MTT assay, and a wound-healing scratch assay and a Transwell invasion assay were conducted to evaluate cell motility and invasion capability, respectively, using the procedure previously described [48 (link)].
7
Candidalysin-mediated platelet activation
8
Hospital Wastewater Concentration Protocol
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Hospital wastewater was collected from the main sewer outflow of a Pittsburgh area hospital. Municipal wastewater was collected from two different municipal wastewater treatment facilities located in the Pittsburgh metropolitan area. Each wastewater sample was centrifuged at 3200 g for 20 min, and the supernatant was filtered using a 0.22 μm filter and concentrated by centrifuging in an Amicon filter unit (MilliporeSigma, Burlington, MA) at 3200 g for 15 min.
9
Magnetic Bead-Conjugated Zinc Finger Protein Purification
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The protein storage buffer was exchanged to ZHEPES buffer (pH 7.5) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.1 mM ZnCl2, 30 mM KCl, 1 mM MgCl2, and 5 mM DTT at pH 7.5 using 10-kDa Amicon filter unit (MilliporeSigma, Burlington, MA) by centrifugation at 4,000 × g for 1 h at 4°C. The ZFP concentration was re-evaluated using a Nanodrop (ThermoFisher, Waltham, MA). The concentrated ZFP was conjugated on Dynabeads M-280 Tosylactivated (ThermoFisher, USA) according to the manufacturer's protocol. The magnetic beads are polystyrene beads coated with a polyurethane layer. 1 mg of the magnetic beads was washed with 35 ml buffer A (0.1 M borate buffer pH 9.5) twice and then incubated 12 -16 h with a mixture of 20 mg of ZFP, 30 ml of buffer A, and 20 ml of buffer C (3 M ammonium sulfate in Buffer B containing 0.1 M sodium phosphate buffer with 0.1 mM ZnCl2, pH 7.4) at 37°C with shaking at 250 rpm. After overnight incubation, the beads were incubated with buffer D (PBS pH 7.4 with 0.5% (w/v) BSA, 5 mM DTT, and 0.1 mM ZnCl2) for 1 h at 37°C with shaking at 250 rpm and washed thoroughly using buffer E (PBS at pH 7.4 with 0.1% (w/v) BSA, 5 mM DTT, and 0.1 mM ZnCl2) twice. Finally, 48 ml of buffer E was added and stored at 4°C until use.
10
Purification and Characterization of rPaAP
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rPaAP expression, purification, and refolding were carried out as described previously (15 (link)), and the refolded protein was concentrated using 10,000-MWCO Amicon filter units (Millipore-Sigma). The concentrated material was then dialyzed using the same method described above, applied to an S200 Sephadex ion exchange column, and eluted with a gradient of 0 to 2 M NaCl, pH 7.6, at a flow rate of 0.02 ml/min, and 18 2-ml fractions were collected. Fractions were assayed for leucine aminopeptidase activity as described previously (10 (link)) and analyzed by SDS-PAGE and Ruby staining as described above. Fractions containing pure rPaAP were pooled and concentrated using Amicon 10,000-MWCO filter units to ∼0.5 μg/ml.
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