What Is the General Procedure for GST Pull-Down Analysis of Protein–Protein Interactions?

GST pull-down is a widely employed method for investigating protein–protein interactions. The principle relies on the specific and high-affinity binding of the GST (Glutathione S-Transferase) tag to reduced glutathione (GSH). In this assay, a bait protein is expressed as a fusion with GST, which facilitates its immobilization on glutathione-coated beads (e.g., glutathione agarose). When a protein mixture containing potential interacting partners (prey proteins) is incubated with the immobilized GST-fusion protein, specific binding partners are retained on the beads. After removal of non-specifically bound proteins through washing, interacting proteins can be eluted and analyzed.

 

The typical workflow of a GST pull-down assay includes the following steps:

 

1. Expression and Purification of Gst-Fusion Protein

The gene encoding the bait protein is cloned into a GST-tag expression vector and introduced into a suitable host system (e.g., Escherichia coli or yeast). Following expression induction, cells are harvested and lysed to release intracellular proteins. The GST-fusion protein is purified by affinity chromatography using glutathione-conjugated beads, followed by thorough washing and elution steps.

 

2. Preparation of Cell or Tissue Lysate

Cells or tissues containing candidate prey proteins are lysed under conditions that preserve native protein–protein interactions. The lysate is clarified by centrifugation to obtain a soluble protein extract for binding reactions.

 

3. Gst Pull-Down Assay

(1) The purified GST-fusion protein is incubated with the prepared cell lysate at 4°C for several hours or overnight to allow binding interactions.

(2) Glutathione-conjugated beads are then added to the mixture and incubated for 1–2 hours. The beads are subsequently collected by centrifugation. The GST-tagged bait protein binds to the beads, and any interacting prey proteins are co-precipitated.

(3) The beads are washed multiple times with wash buffer to remove non-specifically bound proteins. Stringent washing conditions can be applied to improve interaction specificity.

(4) Bound proteins are eluted using a buffer containing excess reduced glutathione, which competes for binding to the GST tag and thereby releases both the GST-fusion protein and associated prey proteins from the beads.

 

4. Identification and Analysis of Interacting Proteins

The eluted protein complexes are resolved by SDS-PAGE and visualized by silver staining or subjected to immunodetection via Western blotting. If a known target is suspected, a specific antibody can be employed for detection. In the absence of prior knowledge about the interacting proteins, protein bands can be excised from the gel and analyzed by mass spectrometry to identify the binding partners.

 

This protocol provides a general framework for conducting GST pull-down assays, though specific steps and conditions may vary depending on experimental design and research objectives.

 

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