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Cell Culture Transfection for Production and Purification of Wnt Ligands
Published: Jan 20, 2012 DOI: 10.21769/BioProtoc.176 Views: 20524
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Abstract
Wnt ligand proteins are extremely difficult to purify and enrich in vitro. This protocol uses Wnt11r protein as an example to illustrate how to use 293T cells to produce secreted Wnt11r and collect it in vitro for further biochemical experiments.
Materials and Reagents
- 293T cells
- 0.1 M glycine-HCl (pH 3.5)
- 0.5 M Tris-HCl
- Fetal bovine serum (FBS) (Life Technologies, Invitrogen™, catalog number: 10438-026 )
- Dulbecco's modified eagle medium (DMEM) (high glucose) (Life Technologies, Invitrogen™, catalog number: 11965 )
- Trypsin EDTA (Life Technologies, Invitrogen™, catalog number: 15050-065 )
- PBS buffer (Life Technologies, Invitrogen™, catalog number: 14040-182 )
- Anti-FLAG M2 (Sigma-Aldrich, catalog number: A2220 )
- Pen/Strep (Sigma-Aldrich, catalog number: P4333 )
- Effectene transfection regent (QIAGEN, catalog number: 301425 )
- Media (see Recipes)
Equipment
- Standard tabletop centrifuges
- 37 °C, 5% CO2 incubator
- Water bath
- 50 ml conical tubes
- Nitrocellulose membrane
- 100 mm Petri dishes
Procedure
Category
Developmental Biology > Cell signaling > Ligand
Cell Biology > Cell isolation and culture > Transformation
Biochemistry > Protein > Fluorescence
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