【爆款】pull-down技术.ppt

GST
(glutathione-sepharose beads) 35S-labled cell lysate
GST
GST Protein X Interact at 40C
Microfuge to collect complexes
GST Protein X
Analyze by SDSPAGE
LanBaidu Nhomakorabea1. Marker Lane2. GST-protein X Lane3. GST
protein with a mutated interaction domain, 4) To test for binding between the putative protein and GST.
Method
Preclearing the cell lysate--- Incubation, 40C, 2h Centrifugation,40C
The proteins are resolved by SDS-PAGE, and processed by Western blotting, autoradiography or protein staining.
GST Protein X
(glutathione-sepharose beads) 35S-labled cell lysate
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;
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autoradiograph
The schema of a GST pull-down experiment
Two general uses :
To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins;
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.
cell lysate + glutathione agarose beads + GST
supernatant
End-over-end mixing
Probing the cell lysate---
Precleared cell lysate + glutathione agarose beads + GST
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;
Incubation, 40C, 2h Centrifugation,40C
Precleared cell lysate + glutathione agarose beads + GST
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.
To confirm suspected interactions between the probe protein and a known proteins.
antibodies to the target protein, or: 1) the 35S-labeled in vitro translated protein, or the target protein can be tagged with
an epitope; 2) Cell in culture can be transfected with a plasmid encoding the target protein to
increase the abundance; 3) To control the specificity of binding, the best is the inclusion of a GST fusion
1) The protein concentrations, 2) Is the probe protein normally expressed in that particular cell or tissue? 3) Is the goal to compare different types of cell populations?