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GST Pull Down Protein Interaction Complex Protein Expression And Purification

GST Pull Down Protein Interaction Complex Protein Expression And Purification
GST Pull Down Protein Interaction Complex Protein Expression And Purification

Large Image :  GST Pull Down Protein Interaction Complex Protein Expression And Purification

Product Details:

Brand Name: CELLFREE
Model Number: CF-PR-010

Payment & Shipping Terms:

Packaging Details: vial
Delivery Time: ~3 weeks
Detailed Product Description
Starting Material: Bait And Target Proteins Or Bacterial Lysates Starting Information: Bait And/or Target Protein/gene Sequence
Deliverables: Cloning Vector,Bait And/or Target Protein Protein Purity: > 90%
Electronic Data: Nteraction Analysis Report Protein Preparation: Optional
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GST Pull Down Protein Expression Purification

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Protein Interaction Complex Purification

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Protein Interaction Complex Expression

Introduction to pull-down assays

 

The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. Pull-down assays are useful for both confirming the existence of a protein–protein interaction predicted by other research techniques (e.g., co-immunoprecipitation) and as an initial screening assay for identifying previously unknown protein–protein interactions.

Pull-down assays are a form of affinity purification and are similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) methodologies greatly enhance the speed and efficiency of protein purification and simultaneously provide the technology platform to perform a pull-down, or co-purification, of potential binding partners. In a pull-down assay, a bait protein is tagged and captured on an immobilized affinity ligand specific for the tag, thereby generating a "secondary affinity support"’ for purifying other proteins that interact with the bait protein. The secondary affinity support of immobilized bait is then incubated with a protein source that contains putative "prey" proteins, such as a cell lysate. The source of prey protein at this step depends on whether the researcher is confirming a previously suspected protein–protein interaction or identifying an unknown interaction. The method of protein elution depends on the affinity ligand and ranges from using competitive analytes to low pH or reducing buffers.

Besides investigating the interaction of two or more proteins, pull-down assays are a powerful tool to detect the activation status of specific proteins. For example, proteins that are activated in response to tyrosine phosphorylation can be pulled down using an immobilized SH2 domain that targets the phosphorylated tyrosine on a given protein. Additionally, GTPases, which act as molecular switches that regulate cell signaling by cycling between a GTP-bound (active) and GDP-bound (inactive) state, can be pulled down using an immobilized GTPase-binding domain of downstream proteins that are recruited to GTP-bound, activated GTPases. In both types of pull-down assays, because the specificity of the interaction is dependent on the sequence of the binding domain, these approaches are highly specific in detecting the activation of distinct proteins.

 

Advantages and disadvantages with different pull-down assays for the retrieval of protein complexes

 

Method Advantages Disadvantages
Affinity pull-down Generic Ability to purify low-abundant protein complexes The presence of a protein tag may influence results Competition with the endogenous complex
Tandem affinity purification (TAP) Generic Ability to purify low-abundant protein complexes Mild conditions used throughout The presence of a protein tag may influence results Competition with the endogenous complex
Co-immunoprecipitation Does not require cloning and heterologous expression. Rapid if antibody is available Not generic - requires access to specific antibodies

 

Pull Down Service content

 

Customer Provides Services Content Deliverables Timeline
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
Bait and/or target protein/gene sequence 4~6 weeks

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Contact Details
CELLFREE SCIENCE CO., LTD.

Contact Person: Pan Lin

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