A standard pull-down assay requires a purified protein with a recombinant affinity tag that acts as 'bait' and is immobilized on a tag-specific affinity ligand. Another protein, or 'prey' protein, is then incubated with the 'bait' protein. When these two proteins physically interact, the 'prey' protein is captured by the 'bait' protein. Subsequently, depending on the affinity ligand, the interacting 'prey' protein can be eluted using an elution buffer. Finally, both the 'prey' and 'bait' proteins can be separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by protein blotting.
The GST pull-down fusion protein sedimentation technique uses genetic recombination to insert a vector with a GST tag into target protein A. Glutathione-coated beads bind to the target fusion protein. After adding cell lysate, interacting proteins are adsorbed by the fusion protein. Excess glutathione is then added for elution. The eluted sample is analyzed and identified using mass spectrometry. The GST pull-down fusion protein sedimentation technique primarily studies strong or stable protein-protein interactions in vitro. It can identify potential direct interactions between two known proteins of interest and find unknown proteins that may interact with the target protein.
In addition to confirming interactions between proteins, the GST pull-down fusion protein sedimentation technique can also be used to characterize the conditions of protein interactions. For example, using domain truncation methods with pull-down techniques can determine functional regions critical for protein interactions. Additionally, this technique can explore chaperones of target protein interactions by incubating the 'bait' protein with a mixture of proteins or cell/tissue extracts. After washing steps, bound proteins can be eluted and identified by mass spectrometry using high-throughput methods.
As a leading omics technology service company, BioTide has years of experience in mass spectrometry-based protein interaction analysis. BioTide offers efficient strategies for protein expression and purification, as well as optimized solutions for pull-down protein interaction analyses. With a focus on strict quality control and shorter testing cycles, BioTide provides customizable, one-stop services to save valuable time and budget, covering every step from gene synthesis to data reporting in your project.
• Gene synthesis, expression plasmid construction - expression, purification, and immobilization of bait proteins - expression and purification of prey proteins - incubation, washing, and elution
• Identification of protein interactions via SDS-PAGE and Western blotting
• LC-MS mass spectrometry identification of interacting proteins - data analysis - issuance of bilingual reports - free consultation
