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A General Protocol for GST Pull-down
Published: Jan 20, 2012 DOI: 10.21769/BioProtoc.177 Views: 75482
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Abstract
GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure.
Materials and Reagents
- pGEX plasmid (GE Healthcare Dharmacon)
- IPTG
- PBS
- Isoproponal
- Ethanol
- TritonTM X-100
- SDS-PAGE
- Tryptone
- NaCl
- NaOH
- Agar
- Tris-HCl
- PMSF
- NaVO3
- NP-40 w
- Sodium-deoxycholate
- Commassive blue staining (Ezblue gel staining reagent) (Sigma-Aldrich, catalog number: G1041 )
- lysozyme (Sigma-Aldrich, catalog number: L7651 )
- Glutathione sepharose 4B (GE Healthcare Dharmacon, catalog number: 17-0756-01 )
- Protease inhibitors (Sigma-Aldrich, catalog number: P2741 )
- Phosphatase inhibitors (Sigma-Aldrich, catalog number: P2850 / P5726 )
- 2x YTA (see Recipes)
- Elution buffer (see Recipes)
- Lysis buffer (see Recipes)
Equipment
- Standard tabletop centrifuges
- Shaker
- Ultra Centrifugal Filter (EMD Millipore, catalog number: UFC900308 )
- Water bath
- Sonicator
Procedure
Category
Biochemistry > Protein > Immunodetection
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