One of the best tools for investigating the function of a protein comes from characterization of its molecular interactions. Co-immmunoprecipitation and Glutathione-S-Transferase (GST) fusion protein pull-down assays are two commonly utilized techniques for probing the molecular interactions of a protein of interest.
GST pull down is a common method for detecting protein interactions in vitro. It can verify the interaction between known proteins and can also be used to screen unknown proteins that interact with known proteins. This method is convenient and simple to operate.
GST pull-down is one of the methods to verify the binding of two proteins in vitro. The principle is that the target protein is fused and expressed with GST, and the protein is immobilized on the glutathione (GSH) affinity resin as a support for affinity with the target protein and acts as a "bait protein". The target protein solution is passed through the column, from which the interacting "capture protein" (target protein) can be captured. The interaction between the two proteins was confirmed by western-blotting (WB) analysis after elution of the conjugate.
Both "bait protein" and "capture protein" can be obtained by cell lysates, purified proteins, expression systems, and in vitro transcription and translation systems. This method is simple and easy to operate.
In vitro GST pull-down assay.
(1) Direct protein-protein interactions can be verified.
(2) GSH glutathione-coupled beads have high affinity and high elution purity.
(3) GST pull-downs use recombinant protein they are more cost-effective.
(4) Detect more specific investigations of protein-protein interactions without contamination from additional protein complex components
(1) The verification of interactions is a biochemical reaction in the test tube, which cannot fully reflect the true interactions of intracellular proteins.
(2) The fusion-expressed GST tag, with a long peptide chain, may change the original folding structure of the original target protein.
Difference between GST Pull-down and Co-IP
GST pull-down: GST fusion protein pull-down assays rely on purified proteins, which may not fully mimic a protein's native conformation or posttranslational modification mediating the interaction. ; Co-IP experiment: intracellular high expression of the target protein through the IgG globule-antibody-protein complex system to isolate the interacting protein complexes. It detects in vivo reactive binding within the cell.
larger quantities of "bait"can be utilized for more sensitive detection of interactions.
As a leading omics technology service company, Creative Proteomics has many years of experience in mass spectrometry-based protein interaction analysis. Creative Proteomics provides efficient strategies for protein expression and purification, as well as optimal optimization solutions for pull-down protein interaction analysis. Featuring strict quality control and short testing cycles, we provide customers with a customizable one-stop service covering every step of your project from gene synthesis to data reporting:
Provide complete experimental report, SDS-PAGE or WB results.
|GST pull-down (target protein and bait protein prokaryotic vector construction)|
|GST pull-down (prokaryotic expression of target protein and bait protein)|
|GST pull-down and WB detection|