Creative Biostructure

Online Inquiry

Please enter "biostructure" as the verification code.

Protein pulldown assay takes advantage of a bait protein for target protein enrichment based on the interaction with the bait protein and target protein. It is an in vitro affinity purification method that can be used to confirm the existing protein-protein interactions, or to identify novel protein-protein interactions. Creative Biostructure provides technical support for protein pulldown as well as other techniques in protein purification, and we also have the most advanced instruments to perform protein pulldown with high-efficiency.

Pulldown assay is very similar to immunoprecipitation, only in which an antibody as the affinity reagent replaces the bait protein. The bait protein are usually with GST-tag, His-tag, or biotin-tag to capture or pulldown the target protein.

General Steps in a GST-tagged Protein Pulldown Assay

  1. Choose the target protein;
  2. Plasmid construction of GST-tagged protein;
  3. Target protein expression;
  4. Target protein purification;
  5. Cross-linking of target protein to beads;   >>more
  6. Preparation of cell lysate or tissue;   >>more
  7. Pulldown;   >>more
  8. Elute proteins for SDS-PAGE analysis;
  9. Mass Spectrometry Analysis;
  10. Confirmation of the pulldown results with methods such as yeast two-hybird, surface plasmon resonance, co-immunoprecipitation, and fluorescence resonance energy transfer, etc.

Troubleshooting:

1. Interacting protein was not isolated.
If it dues to weak or transient interactions, you may decrease the ion concentration of the buffer or wash time;
If it dues to low-level expression of prey protein, please adjust your culture condition and increase the amount of the target protein.

2. Get large amount of nonspecific proteins after pulldown.
It may caused by the adhesion of hydrophobic proteins to the beads, you can add detergent such as Triton X-100 in the wash buffer to thoroughly wash the beads.

Besides pulldown, Creative Biostructure is also able to help your protein purification project with other technical resources. Please see the table below for more information and feel free to Contact Us.


Related Sections

Services: