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. Bacterially expressed glutathione S-transferase (GST)-fused proteins are used as probes to perform direct measure of protein-protein interactions and for affinity purification.

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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

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

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.


Detection of protein protein interactions using the gst fusion protein pull down technique

  • 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.


Detection of protein protein interactions using the gst fusion protein pull down technique

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


Detection of protein protein interactions using the gst fusion protein pull down technique

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


Detection of protein protein interactions using the gst fusion protein pull down technique

  • 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.


Method

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) .


Detection of protein protein interactions using the gst fusion protein pull down technique

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

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.


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