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GST Pull-Down Service

What is GST Pull-Down?

The GST pull-down assay is a powerful biochemical technique used to investigate protein-protein interactions (PPIs), which are essential for understanding cellular processes and signaling pathways. In this assay, a target protein of interest (the "bait") is genetically fused with Glutathione S-Transferase (GST), a protein that has a strong affinity for glutathione. The resulting GST-tagged bait protein is then immobilized on a solid support (such as glutathione-coated beads), creating a platform for capturing any prey proteins that interact with the bait. When incubated with a sample, such as cell lysates, the bait protein binds to its interacting partners, pulling them down from the mixture. The captured proteins are then eluted and identified through downstream analyses, such as SDS-PAGE, Western blotting, or mass spectrometry (MS). This method allows researchers to discover novel protein interactions, validate predicted interactions, and explore the dynamics of multiprotein complexes.

Creative Proteomics, a leader in proteomics services for over 20 years, is proud to offer our state-of-the-art GST pull-down service. Our platform is designed for the in-depth analysis of PPIs, enabling researchers to elucidate complex cellular networks with exceptional precision and reproducibility. Through our advanced methodology and rigorous quality control, we help drive groundbreaking discoveries in cellular biology, disease mechanisms, and drug development.

Outline of the GST pull-down.

Figure 1. Schematic of the GST pull-down assay procedure.

What is the Difference Between GST Pull-Down and Co-IP?

Both GST pull-down and co-immunoprecipitation (Co-IP) are pivotal in the study of PPIs. However, they differ in their fundamental approach and requirements. The table below summarizes these differences:

Feature GST Pull-Down Co-Immunoprecipitation (Co-IP)
Principle Utilizes a GST-tagged bait protein bound to glutathione beads for affinity capture of interactors. Employs specific antibodies to precipitate the target protein along with its binding partners.
Specificity High specificity due to the strong and selective GST–glutathione interaction. Dependent on the specificity and quality of the antibody used; may encounter higher background.
Reagents Required Requires expression and purification of a GST-fusion protein. Requires high-quality, specific antibodies against the protein of interest.
Workflow Complexity Relatively straightforward with controlled affinity binding conditions. Can be more complex due to variable antibody performance and optimization needs.
Detection Methods Downstream analysis via SDS-PAGE, Western blot, and mass spectrometry. Similar detection techniques, but results are antibody-dependent.

Advantages of GST Pull-Down Technology

Creative Proteomics' GST Pull-Down Service Workflow

Creative Proteomics' GST pull-down service workflow.

Applications of GST Pull-Down Service

Simple Requirements for GST Pull-Down

Requirement Purified Protein Plant and Animal Tissues Cell Lysis Solution
Volume and Concentration 3-5 mg of purified protein with a concentration of ≥1 mg/mL 10-20 mg of tissue per assay. 1 mL of lysis buffer per 10 mg of tissue
Purity ≥90% purity, as determined by SDS-PAGE or HPLC N/A (tissues are the starting material) Ensure the buffer is free from proteases and nucleases
Storage -80°C in aliquots to prevent freeze-thaw cycles Flash freeze in liquid nitrogen and store at -80°C Store at 4°C for short-term use; aliquot and store at -20°C for long-term storage
Shipping Dry ice to maintain protein stability Dry ice to preserve tissue integrity. 4°C to maintain buffer integrity

FAQ

Q1: How do I ensure that the GST tag doesn't interfere with the function or folding of my bait protein?

A1: The GST tag is typically placed at the N- or C-terminal end of the bait protein to minimize interference with its functional domains. If you're concerned about interference, we can offer advice on using smaller or removable tags, or you could use a cleavable linker to remove the tag after the pull-down process.

Q2: What are the limitations of GST pull-down assays?

A2: While effective, GST pull-down assays have a few limitations:

GST tag interference: The large GST tag may affect the folding or function of the protein of interest.

Weak or transient interactions: Some interactions may be missed due to weak binding or transient nature.

Requirement for purified protein: The success of the assay depends on having sufficiently pure bait proteins for efficient interaction capture.

Q3: Can GST pull-down assays be used to study protein interactions in a membrane-bound environment?

A3: Yes, GST pull-down assays can be adapted to study membrane proteins. However, you may need to optimize the lysis buffer to retain membrane protein integrity, as well as use detergents that help solubilize membrane-bound proteins without disrupting interactions.

Q4: What steps do you take to ensure that the GST pull-down assay results are not affected by contaminants or non-specific binding?

A4: We implement multiple measures to minimize contaminants, such as:

Using stringent washing conditions to remove non-specifically bound proteins.

Employing control experiments (e.g., using untagged fusion proteins or blank beads) to detect any background interference.

Including a negative control sample to help validate the specificity of the protein interactions.

Demo

Demo: TRAM Is Involved in IL-18 Signaling and Functions as a Sorting Adaptor for MyD88.

GST pull-down assay for protein interaction.

Figure 2. Protein interaction assays. (A) GST pull-down assay investigating the direct interactions between the GST-MyD88-TIR and the TRAM-TIR or TLR1-TIR. (B) GST pull-down assay between the GST-IL-18 receptor TIRs and TRAM-TIR. (Ohnishi H, et al., 2012)

Case Study

Case: The ARH Adaptor Protein Regulates Endocytosis of the ROMK Potassium Secretory Channel in Mouse Kidney

Abstract: Renal outer medullary potassium (ROMK) channels are essential for maintaining potassium balance in the kidney. They are regulated by factors such as plasma potassium levels, aldosterone, and dietary intake. ROMK channels are constitutively active, and their function is modulated by their presence at the apical membrane of renal cells. The internalization of ROMK channels through clathrin-dependent endocytosis is crucial in regulating potassium excretion, especially during potassium deficiency. Aberrant ROMK endocytosis may contribute to hyperkalemia in renal diseases. The mechanism of ROMK internalization remains poorly understood, though previous studies suggest the involvement of WNK kinases and clathrin adaptors. This study focuses on identifying the adaptor protein ARH (autosomal recessive hypercholesterolemia) as a key regulator of ROMK endocytosis.

Methods

GST Pull-Down: To identify interacting proteins, GST-fusion ROMK C-terminal regions were incubated with His-tagged PTB-CLASP adaptor proteins. Ni-NTA beads were used to capture His-tagged proteins, and after washing, bound proteins were analyzed by SDS-PAGE and western blotting.

Co-Immunoprecipitation: Protein-protein interactions between ARH and ROMK were examined through immunoprecipitation using anti-ARH antibodies. This method enabled the isolation and analysis of ROMK and ARH complexes from kidney extracts.

Western Blotting: To detect specific proteins, the immunoprecipitated proteins were transferred to nitrocellulose membranes and probed with antibodies against GST or His tags, followed by HRP-conjugated secondary antibodies.

Results

ARH Binds to ROMK: ARH specifically binds to ROMK through a novel internalization signal in ROMK's C-terminal region, YxNPxFV. This interaction was confirmed through affinity chromatography and immunoprecipitation.

ARH and ROMK Colocalization in the Kidney: In kidney cells, ARH and ROMK were observed to colocalize in the distal nephron, particularly in areas involved in potassium regulation. Co-immunoprecipitation studies showed that ARH interacts specifically with ROMK in kidney tissue.

ARH Promotes ROMK Endocytosis: In COS-7 cells, the presence of ARH increased the internalization of ROMK channels. Surface biotinylation assays revealed that ARH expression led to a significant reduction in ROMK on the cell surface, indicating that ARH promotes ROMK endocytosis.

Effect of Mutations on ARH Binding and Endocytosis: Mutating the NPNF motif in ROMK blocked the interaction with ARH and prevented the endocytosis of ROMK, confirming that this motif is crucial for ARH-mediated internalization.

ARH in Response to Potassium Levels in Mice: In ARH knockout mice, the regulation of ROMK protein in response to dietary potassium was impaired. In wild-type mice, a low-potassium diet reduced ROMK levels, but in ARH knockout mice, ROMK levels remained unchanged regardless of potassium intake.

ARH in WNK1 Pathway: The study also showed that L-WNK1, a long isoform of WNK1, stimulates ROMK endocytosis in an ARH-dependent manner, highlighting ARH's role in the WNK1 signaling pathway.

ROMK interacts with ARH.

Figure 3. ROMK preferentially interacts with ARH. (A) Illustration of the pull-down assay. (B) GST fusions of the WT ROMK1 C terminus (ROMK-C) (amino acids 349–391), LRP C terminus (LRP-C), and His-tagged PTB proteins were purified to homogeneity for the binding assay. (C) Binding assay. (D and E) ARH interacts with ROMK in an NPXF signal–dependent manner.

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