pull-down技术ppt课件
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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.
Two general uses :
To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins;
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?
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.
protein with a mutated interaction domain,
4) To test for binding between the putative protein and GST.
6
MLeabharlann Baiduthod
Preclearing the cell lysate--- Incubation, 40C, 2h Centrifugation,40C
Microfuge to collect complexes
4
GST Protein X
Analyze by SDSPAGE
Lane1. Marker Lane2. GST-protein X Lane3. GST
123
autoradiograph
The schema of a GST pull-down experiment 5
3
GST Protein X
(glutathione-sepharose beads) 35S-labled cell lysate
GST
(glutathione-sepharose beads) 35S-labled cell lysate
GST
GST Protein X Interact at 40C
Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique
1
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
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
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
Incubation, 40C, 2h Centrifugation,40C
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; 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.
Two general uses :
To identify novel interactions between a fusion (or probe) protein and unknown (or target) proteins;
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?
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.
protein with a mutated interaction domain,
4) To test for binding between the putative protein and GST.
6
MLeabharlann Baiduthod
Preclearing the cell lysate--- Incubation, 40C, 2h Centrifugation,40C
Microfuge to collect complexes
4
GST Protein X
Analyze by SDSPAGE
Lane1. Marker Lane2. GST-protein X Lane3. GST
123
autoradiograph
The schema of a GST pull-down experiment 5
3
GST Protein X
(glutathione-sepharose beads) 35S-labled cell lysate
GST
(glutathione-sepharose beads) 35S-labled cell lysate
GST
GST Protein X Interact at 40C
Detection of Protein-Protein Interactions Using the GST Fusion Protein Pull-down Technique
1
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
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
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
Incubation, 40C, 2h Centrifugation,40C
Precleared cell lysate + glutathione agarose beads + GST