GST Pull-Down Service

Inquiry

CD BioSciences has extensive experience in plant protein research and provides GST pull-down assay to help clients verify protein-protein interaction. We use specific secondary antibodies to avoid signal interference from antibody-heavy chains and significantly improve the detection results.

GST pull-down is based on the fact that glutathione-S-transferase (GST) can bind to Glutathione (GSH). Therefore, GST-bait protein can bind to GSH-agarose beads, and if prey proteins that interact with the bait protein are present in the environment, an "agarose bead-GSH-GST-bait-prey" complex will be formed. After elution, the interaction can be analyzed based on SDS-PAGE and Western Blot.

Figure 1. In vitro GST pull-down assay. (Bernhard, O. K., et al., 2005)

Service Content

Experimental content

GST-bait protein prokaryotic expression vector construction (optional).
Bait protein expression and Western Blot detection.
Cross-linking of bait protein with magnetic beads.
Prey protein expression and Western Blot detection.
GST pull-down capture of bait proteins with interaction proteins, including incubation, washing, and elution. (experimental group and empty control group)
Identification of protein-protein interaction by SDS-PAGE and Western Blot.

Bait labels we can provide

Bait labels	Affinity ligands
Glutathione S-transferase (GST)	Glutathione
His-tag	Nickel or cobalt chelate
Biotin	Streptavidin

GST pull-down optimization service

Optimizing binding conditions

We help clients screen appropriate buffers and conditions to improve the binding efficiency and specificity of prey proteins to GST-bait proteins, including optimizing pH, ionic strength, temperature, and binding time.

Preventing non-specific binding

To minimize interference from non-specific binding, we provide non-specific proteins (BSAs) or peptides with competitive binding as blockers.

Selection of enzyme inhibitors

If protein-protein interactions are affected by specific enzymes, we can provide clients with the appropriate enzyme inhibitors to prevent enzyme interference.

Advantages and disadvantages of GST pull-down technology

Advantages	Disadvantages
Validation of direct protein-protein interactions. GSH-coupled beads have high affinity and high elution purity. More cost-effective using recombinant proteins. Detect more specific studies of protein-protein interactions without contamination by other protein complex components.	It cannot fully reflect the real interactions of intracellular proteins. Fusion-expressed GST tags have long peptide chains that may alter the folding structure of the original target protein.

Materials to be Provided by Clients

Gene sequences of bait and prey proteins.

Expression plasmid, which needs to provide the sequencing peak graph.

Protein samples, lysate containing bait and prey proteins > 5ml.

Immunoassay antibody > 20ul.

Delivery Reports

Constructed cloning vector.
Bait and interaction proteins 3-5mg (purity > 90%).
Interaction analysis report (SDS-PAGE, silver staining, Western Blot).
Experimental procedure report with original pictures and data.

CD BioSciences is a biological company specializing in plant protein research, providing protein interaction assays including GST pull-down. If you need to research protein interactions, please contact us, we have deep expertise in plant molecular biology technology, with cutting-edge facilities and methods to help you further investigate protein signaling, functional proteomics, and protein complex dynamics in plants.

Reference

Bernhard, O. K., et al. (2005). New insights into viral structure and virus-cell interactions through proteomics. Expert review of proteomics. 2(4), 577–588.

For research use only, not for clinical use.