- Home
- Protocols
-
A General Protocol for GST Pull-down
Published: Jan 20, 2012 DOI: 10.21769/BioProtoc.177 Views: 75502
PDF 
Ask a question
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
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Post a Question
