pull-down技术.pptVIP

The GST fusion protein probe is expressed and purified from bacteria; In parallel, a cell lysate (which can be 35S-labled or unlabled) is prepared; The GST fusion protein probe and the cell lysate are mixed, in the presence of glutathione-agarose beads and incubate the mixture to allow protein associations to occure; By centrifugation, collect the GST fusion probe protein and any associated molecules; The complexes are washed and can be eluted from the beads with excess free glutathione or boiled directly in SDSbuffer; The proteins are resolved by SDS, and processed by Western blotting, autoradiography or protein staining. Method To optimize the GST pull-down technique for each protein complex : The buffer (such as RIPA )in which the interactions take place; The amount of target protein mixed with the fusion(probe) protein; The condictions used to wash the beads to eliminate nonspecific interactions. Troubleshooting GST pull-down experiment  * * Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique Bacterially expressed glutathione S-transferase (GST)-fused proteins are used as probes to perform direct measure of protein-protein interactions and for affinity purification. The GST Fusion protein pull-down technique (Kaelin et al.1991) uses the affinity of GST for glutathione-coupled beads to purify interacting proteins from a solution of non interacting proteins. GST Protein X GST 35S-labled cell lysate (glutathione-sepharose beads) (glutathione-sepharose beads) 35S-labled cell lysate GST Protein X GST GST Interact at 40C Microfuge to collect complexes 1 2 3 autoradiograph Analyze by SDSLane1. Marker Lane2. GST-protein X Lane3. GST The schema of a GST pull-down experiment GST GST Protein X Two general uses : To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins; The protein concentrations, Is the p