GST Pull-Down Assays Service

Many proteins function in association with partner proteins or as components of large multiprotein complexes. Understanding these protein interactions is critical to our understanding of biological pathways and cellular function.

The principle of pull-down assay is affinity purification, which is similar to immunoprecipitation, except that this method uses bait protein, instead of antibody to trap the target protein. In a pull-down assay, a bait protein is tagged and immobilized on beads with affinity ligand specific for the tag, thereby generating a "secondary affinity support" for purifying other proteins that interact with the bait protein. When incubated with a protein sample that contains putative "prey" proteins, such as a cell lysate, the bait can bind the prey, and thus trap down the prey from the lysate.

There are many types of tags for the bait of pull-down assay, such as GST, poly-His, and biotin, etc. The affinity ligand used to immobilize bait are glutathione, nickel and chelate complexes, and streptavidin, respectively.

Bait tag	Affinity ligand
Glutathione S-transferase (GST)	Glutathione
Poly-histidine	Nickel or cobalt chelate complexes
Biotin	Streptavidin

Service Content

We will elaborately design our experimental scheme according to the protein of your interest, for example, its structure and function domain, cellular function, enzymatic or other activity, etc.

Our GST Pull-Down Assay service process mainly includes Experiment design based on your specific project needs (buffers, beads, tag of bait, etc); Parameters optimization, such as binding time; Cell lysis, pull-down assay(binding, washing, and elution); WesternBlot/ mass spectrometry

Customer Provides	Services Content	Deliverables	Timeline	Price
Bait and target proteins or bacterial lysates	Protein preparation (Optional) Pull-Down assay	Cloning vector 3-5 mg Bait and/or target protein (purity > 90%) Interaction Analysis Report(SDS-PAGE, Silver Staining, Western Blot/MS) Process Report	~3 weeks	Please inquire
Bait and/or target protein/gene sequence			4~6 weeks

Why Choose Us

• Reliable experimental data:

Multiple control groups are set up to eliminate the influence of random factors. The experiment is repeated two times to ensure the validity of the experimental results.

• Perfect experimental facilities:

We have complete downstream supporting facilities, and quantitative analysis can be further carried out by BLI/MS.

• Low background, no stray band: Using SDS-PAGE silver staining, the sensitivity is 100 times higher than that of Coomassie Blue Staining, and the electrophoresis results are clearer.
• Competitive pricing:

It is equivalent to purchasing a kit that guarantees the success of the experiment.

Process of GST Pull Down Assay

GST pull-down is becoming an important tool for validation of suspected protein: protein interactions or for discovering novel protein interactions. GST pull-down uses a GST-fusion protein (bait) bound to glutathione (GSH)-coupled particles to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution. Bait and prey proteins can be obtained from multiple sources including cell lysates, purified proteins, expression systems and in vitro transcription/translation systems.

Troubleshooting Guides

1. Possible cause: Very large amount of GST recombinant protein used as bait.
   Possible solution: Cross-link the GST fusion protein to the GSH beads, or reduce the amount of GST fusion protein used.

2. No specific binding proteins are observed for the protein of interest relative to control.

Possible cause A: Bacterially produced GST fusion proteins are not correctly folded or post- translationally modified.
Possible solution A: Change expression system according to literature on protein of interest and on bacterial expression systems, or use a nonbacterial expression system, for example, in vitro translation, as long as sufficient GST fusion protein for pull-down can be obtained.
Possible cause B: Tissue source for lysate inappropriate for protein of interest.
Possible solution B: Change tissue source: use a tissue that is known to express the protein of interest, or screen several tissues and analyze simultaneously by SDS-PAGE, to compare binding proteins from different tissues; scale up chosen tissue by 10-fold; try tissues isolated from animals of different developmental stages.
Possible cause C: Washing or binding buffers may be too stringent (salt, detergent concentration interferes with binding).
Possible solution C: Dilute out salt or detergent further; homogenize pellets n smaller volumes, or use buffers for tissue extraction that have different properties.

3. Possible solution: Wash the beads with Triton X-100 containing wash buffer, then thoroughly wash the beads again with Triton X-100 free wash buffer.

4. Interacting protein was not isolated.

Possible cause A: Weak or transient interactions.
Possible solution A: Decrease wash times or decrease the ion concentration of the buffer.
Possible cause B: Low expression level of prey protein.
Possible solution B: Increase the amount of prey protein.

Reference:

Thery C, Amigorena S, Raposo G, et al. Current protocols in cell biology[M]. John Wiley, New York, 2006.

Contact Us