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Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique PowerPoint Presentation
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Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique

Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique

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Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique

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  1. Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique

  2. 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.

  3. 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 SDS-PAGE buffer; • The proteins are resolved by SDS-PAGE, and processed by Western blotting, autoradiography or protein staining.

  4. GST Protein X GST (glutathione-sepharose beads) (glutathione-sepharose beads) 35S-labled cell lysate 35S-labled cell lysate GST Interact at 40C GST Protein X GST Microfuge to collect complexes

  5. GST Protein X GST Analyze by SDS-PAGE Lane1. Marker Lane2. GST-protein X Lane3. GST 1 2 3 autoradiograph The schema of a GST pull-down experiment

  6. Two general uses : • To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins; • The protein concentrations, • Is the probe protein normally expressed in that particular cell or tissue? • Is the goal to compare different types of cell populations? • To confirm suspected interactions between the probe protein and a known proteins. • antibodies to the target protein, or: • the 35S-labeled in vitro translated protein, or the target protein can be tagged with an epitope; • Cell in culture can be transfected with a plasmid encoding the target protein to increase the abundance; • To control the specificity of binding, the best is the inclusion of a GST fusion protein with a mutated interaction domain, • To test for binding between the putative protein and GST.

  7. Method • Preclearing the cell lysate--- cell lysate + glutathione agarose beads + GST • Probing the cell lysate--- • Detecting interacting proteins Incubation, 40C, 2h Centrifugation,40C supernatant End-over-end mixing Precleared cell lysate + glutathione agarose beads + GST Incubation, 40C, 2h Centrifugation,40C Precleared cell lysate + glutathione agarose beads + GST fusion probe protein End-over-end mixing optional Wash the beads 3 times Mix the beads or the eluted protein with SDS-PAGE gel loading buffer Elute the GST fusion protein, any proteins boiling SDS-PAGE analysis the samples Detecting (Western blotting, autoradiography or protein staining) .

  8. 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

  9. The GST pulldown is a related technique in which either 1) a single defined in-vitro-expressed protein, 2) an unknown protein present in a pool of proteins in a cell lysate, or 3) an unknown protein expressed from a pool of in-vitro-translated cDNAs is collected by its interaction with a fusion protein composed of the target protein linked to a GST moiety. This technique can also be used to derive semiquantitative estimates of the affinity of protein-protein interactions.