Antibodies were expressed in FGE-expressing Expi293 cells (a generous gift from Melissa Gray) following Thermo Fischer Scientific’s Expi293 expression protocol. 1 μg (0.5 μg heavy chain and 0.5 μg light chain encoding plasmid) of DNA per mL culture was used for transfection. Antibodies were harvested after 7 days by collecting the supernatant from centrifugation at 300g for 5 min, then centrifugation at 3200g for 30 min at 4 °C. The supernatant was filtered using a 0.22 μm filter unit (Fischer Scientific). Antibodies were purified using Protein A-Sepharose® 4B (Thermo Fisher Scientific) column chromatography in dark. The Protein A beads were packed into Econo-Pac chromatography column (BioRad). The beads were then washed with elution buffer (100 mM Glycine in MQ Water, pH 2.8) and equilibrated under PBS. The filtered supernatant was run through the Protein A column 3 times, washed with PBS 3 times, and eluted with elution buffer into a falcon tube containing 100 μl 1 M Tris buffer, pH 8. Antibody was then buffer exchanged to citrate buffer (50 mM sodium citrate, 50 mM NaCl, pH 5.5) using 30 kDa Amicon Centrifugal Filter.
0.22 μm filter unit
Manufactured by Thermo Fisher Scientific
The 0.22 μm filter unit is a laboratory filtration device designed to remove particulates, bacteria, and other microorganisms from liquids. It features a 0.22 μm pore size membrane that effectively retains particles larger than the specified size. This filter unit is commonly used to sterilize and clarify various solutions in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Protein a sepharose 4b, by Thermo Fisher Scientific (2 mentions)
Econo pac chromatography column, by Bio-Rad (2 mentions)
Micro bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti cd63, by BioLegend (1 mentions)
Anti cd9, by Santa Cruz Biotechnology (1 mentions)
Anti cytochrome c, by BD (1 mentions)
3 protocols using 0.22 μm filter unit
1
Antibody Expression and Purification
2
Antibody Expression and Purification
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Antibodies were expressed in FGE-expressing Expi293 cells (a generous gift from Melissa Gray) following Thermo Fischer Scientific’s Expi293 expression protocol. 1 μg (0.5 μg heavy chain and 0.5 μg light chain encoding plasmid) of DNA per mL culture was used for transfection. Antibodies were harvested after 7 days by collecting the supernatant from centrifugation at 300g for 5 min, then centrifugation at 3200g for 30 min at 4 °C. The supernatant was filtered using a 0.22 μm filter unit (Fischer Scientific). Antibodies were purified using Protein A-Sepharose® 4B (Thermo Fisher Scientific) column chromatography in dark. The Protein A beads were packed into Econo-Pac chromatography column (BioRad). The beads were then washed with elution buffer (100 mM Glycine in MQ Water, pH 2.8) and equilibrated under PBS. The filtered supernatant was run through the Protein A column 3 times, washed with PBS 3 times, and eluted with elution buffer into a falcon tube containing 100 μl 1 M Tris buffer, pH 8. Antibody was then buffer exchanged to citrate buffer (50 mM sodium citrate, 50 mM NaCl, pH 5.5) using 30 kDa Amicon Centrifugal Filter.
3
Isolation and Characterization of Extracellular Vesicles
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A375 and A375SM cells (3 × 106 cells) were cultured on a 15 cm-cell culture dish for 48 h in 20 mL of MEM supplemented with 10% EV-depleted FBS. EV-depleted FBS was prepared by ultracentrifugation at 110,000×g using Beckman 45Ti rotor for 70 min at 4℃. HMVECs were cultured for 48 h in EBM2 supplemented with 5% EV-depleted FBS. The CM was centrifuged at 2000×g for 10 min at 4 ℃, and the supernatant was filtered through a 0.22 μm filter unit (Thermo Scientific Nalgene) to thoroughly remove the cellular debris. Then, to prepare EVs, the supernatant was ultracentrifuged (110,000×g, 70 min at 4 ℃) using Beckman 45Ti rotor, the pellet was washed twice with phosphate buffered saline (PBS) by ultracentrifugation at 110,000×g using Beckman 45Ti rotor for 70 min at 4 ℃, and EVs were eluted in 200 μL of PBS. The Micro bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, MA, USA) was used to measure the protein concentration. The isolated EVs were characterized through nanoparticle tracking analysis (Nanosight: Quantum Design, Japan) and western blot procedure with anti-CD9 (1:500, Santa Cruz, #59140), anti-CD63 (1:500, Biolegend, #353013), anti-Cytochrome C (1:500, BD Pharmingen, #556433) antibodies, and anti-Mouse Anti-Hsp70 (1:1000, BD Pharmingen, #610607).
We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV210167)34 (link).
We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV210167)34 (link).
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