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 for GST pull-down assay

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 pull down assay (GST-tagged protein as an example)

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.

GST-pull-downs

Case Study

The experiment was conducted to detect the interaction of protein D- protein B by GST Pull-down assay.

GST-pull-down-case-study
Figure. SDS-PAGE and silver staining analysis

lane 1: GST tag, 7 ul; lane 2: GST Beads + GST tag, 15 ul; lane 3: GST Beads + GST tag + Protein D Flow-through, 10 ul; lane 4: GST Beads + GST tag + Protein D Washed, 10 ul; lane 5: GST Beads + GST tag + Protein D Eluate, 15 ul; lane 6: Protein B (GST tag), 2 ul; lane 7: GST Beads + Protein B (GST tag), 15 ul; lane 8: GST Beads + Protein B (GST tag) + Protein D Flow-through, 10 ul; lane 9: GST Beads + Protein B (GST tag) + Protein D Washed, 10 ul; lane 10: GST Beads + Protein B (GST tag) + Protein D Eluate, 15 ul; lane 11: Marker, 6 ul; lane 12: Protein D, 8 ul; lane 13: GST Beads + Protein D Flow-through, 10 ul; lane 14: GST Beads + Protein D Washed, 10 ul; lane 15: GST Beads + Protein D Eluate, 15 ul

Troubleshooting Guides

Question 1 (Q1): In MS analysis, many or all bands are identified as GST.

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.

Q2: Intense staining of four to five proteins of 20-30 KDa on SDS-PAGE gel, which interfere with the detection of other proteins.

Possible cause: Very large amount of endogenous tissue GST present in the tissue lysate, binding to the remaining free glutathione sites of the GSH beads.

Possible solution: Rigorously pre-clear lysate with GSH beads; if the GST fusion protein is cross-linked to the beads, wash the beads 2 to 3 times to specifically elute the GSTs.

Q3: 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.

Q4: Large amount of nonspecific proteins are eluted from GST fusion protein beads after pull-down.

Possible cause: Hydrophobic (largely membrane) proteins may adhere to the beads when washed without detergent.

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.

Q5: There is still GST fusion protein eluted from the beads after chemical cross-linking, which interferes with pull-down experiment.

Possible cause A: The cross-linking reaction was incomplete or inefficient, or cross-linker has degraded.

Possible solution A: Ensure that sufficient cross-linker is used; requantitate the level of protein bound to GSH beads; cross-link for longer durations (up to 2 h); use fresh cross-linker; switch to more soluble cross-linker.

Possible cause B: GSH washing to remove small amount of non-cross-linked fusion protein was inadequate, or inefficient.

Possible solution B: Wash the column after cross-linking with bead recycling buffer, and allow the solution to pass through.

Q6: 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.

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