What is a GST pull down?
GST pull-down assays involve affinity purifications of one or several unknown proteins from a biological sample using a GST-tagged bait protein. The basic principle is that the GST-tagged bait protein binds to its partners, and the resulting complex is captured on beads with immobilized glutathione.
What is the role of GST in protein purification?
The GST tag Protein purification with affinity tags such as glutathione S-transferase (GST), histidine (HIS), and other affinity tags, enables purification of proteins with both known and unknown biochemical properties.
What is a protein pull down?
The pull-down assay is an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners.
How do you remove GST from protein?
Thrombin or Factor Xa can be removed from the protein of interest in one step using a HiTrap™ Benzamidine FF (high sub) column in series after the GSTrap™ column. In this process, the cleaved, tagged protein and thrombin or Factor Xa is washed from the GSTrap™ column onto the HiTrap™ Benzamidine FF (high sub) column.
How is a pull-down assay done?
In a typical pull-down assay, the immobilized bait protein is incubated with a cell lysate, and after the prescribed washing steps, the complexes are selectively eluted using competitive analytes or low pH or reducing buffers for in-gel or western blot analysis.
How does a pull down assay work?
The basic principle of pull down assay is to utilize a tag fused protein (such as GST-tag, His-tag and biotin-tag) immobilized to affinity resin as the bait protein. Proteins binding to the bait protein (prey protein) can be captured and “pulled down” when the target protein or cell lysate flows through.
How do you elute protein from GST beads?
To elute the protein from the GST tag and agarose bead, add 10µl of thrombin (10 units) per mg GST tagged protein. 2. Mix gently and incubate at room temperature for 2-16 hours.
Does imidazole inhibit thrombin?
Look for Thrombin Sepharose Beads. First you should dialyse your protein into a buffer that is suitable for thrombin, as the Ni-NTA elution buffer is not be the best choice (high salt, reducing agents and imidazole reduce the activity of thrombin).
How does a protein pull down assay work?
The pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. The secondary affinity support of immobilized bait is then incubated with a protein source that contains putative “prey” proteins, such as a cell lysate.