pull-down技术ppt
合集下载
相关主题
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
-
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
-
Bacterially expressed glutathione S-transferase (GST)-fused proteins are used as probes to perform direct measure of protein-protein interactions and for affinity purification.
-
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?
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
Microfuge to collect complexes
-
GST Protein X
Analyze by SDSPAGE
Lane1. Marker Lane2. GST-protein X Lane3. GST
123
autoradiograph
The schema of a GST pull-down experiment
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 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.
Incubation, 40C, 2h Centrifugation,40C
Precleared cell lysate + glutathione agarose beads + GST
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; 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.
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
-
Bacterially expressed glutathione S-transferase (GST)-fused proteins are used as probes to perform direct measure of protein-protein interactions and for affinity purification.
-
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?
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
Microfuge to collect complexes
-
GST Protein X
Analyze by SDSPAGE
Lane1. Marker Lane2. GST-protein X Lane3. GST
123
autoradiograph
The schema of a GST pull-down experiment
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 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.
Incubation, 40C, 2h Centrifugation,40C
Precleared cell lysate + glutathione agarose beads + GST
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; 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.