GST Pull-Down

GST Pull-Down is a powerful technique which studies protein-protein interactions in vitro, allowing the identification of unknown proteins that interact with known proteins and confirmation of suspected interactions, as well as identification of interactions between two known proteins.

The principle of GST Pull-Down is affinity purification with bait protein instead of antibody. The bait protein is tagged with GST by recombinant technology to form a fusion protein which can be bound to GTH (Glutathione) on the solid support through GST due to affinity attraction. When a protein sample, such as cell lysates, passing through the column or mixed with the solid phase complex, the contained putative "prey" proteins that interact with fusion proteins are adsorbed and separated from sample.

• Constructing GST-tagged protein prokaryotic expression plasmid by recombinant technology
• Expressing the fusion protein with GST tag by prokaryotic expression system
• Obtaining high purity fusion protein via the GST affinity purification column and using the GST affinity purification column for protein-to-protein interaction detection
• Eluting target protein
• Separating target protein by SDS-PAGE
• Using WB and LC-MS detection to verify the interaction between proteins or screen for target proteins

Fig 1. Experimental Procedures of GST Pull-Down Assay

• Demonstrate possible interactions between two known proteins
• Discover unknown protein that interact with known proteins
• Extract protein from in vitro transcription/translation lysates

GST Pull-Down is used to identify protein interactions in vitro, it offers a number of advantages.

• Easy to operate
• Identify the direct interactions between protein and protein.

Laminar Flow Hoods

Laminar Flow Hoods are mostly enclosed systems that use filters and directrional air flow to provide a contaminant-free work area for applications requiring a sterile work space, such as tissue culture, culture media preparation, and so on.

Laboratory Shaking Incubators

In order to provide optimal conditions for cell growth, some type of agitation or shaking is necessary to incorporate oxygen and evenly distribute nutrients throughout the culture media. Laboratory Shaking Incubators combine the function of shaker and incubator to provide precise temperature control and simultaneous shaking required by growth of any kind of cell including bacterial cultures, tissue cultures, and yeast.

Refrigerated Microcentrifuges

Refrigerated Microcentrifuge is used to spin small liquid samples at high speeds and low temperature. Centrifugation of small samples is important for many biological applications, such as isolating proteins from solution for use in western blot or 2D electrophoresis.

Electrophoresis

SDS-PAGE using electrophoresis equipment is used to separate out all the proteins according to their molecular within a sample prior to blotting.

Nitrocellulose Membrane

Nitrocellulose membrane refers to a sticky membrane used for immobilizing nucleic acids and proteins. It is a common matrix used in Western blotting due to the high protein-binding affinity. Nitrocellulose membranes are available in 100% pure forms of nitrocellulose with a high surface area, which is uniform. One of the main advantages of nitrocellulose membranes is its very low background as it can be easily blocked.

Gel Imaging Systems

Gel imaging or gel documentation (gel doc) systems are used for the analysis of proteins, antibodies and nucleic acid immobilized in polyacrylamide or agarose gels, membranes or microarrays. Systems come in a variety of configurations depending on throughput and sample type. It is widely used in: monoclonal and polyclonal antibody binding affinities, gel and blot imaging, colony counting, immunoassay, multiplex protein detection and other fields.

Spectrophotometers

Spectrophotometer is an optical instrument that measures the intensity of light in relation to the wavelength. It can measure just about anything such as: plastics, paper, liquid, metal, fabric and so on. A spectrophotometer is used to check for the quality of product in a production facility and identify and quantify microscopic samples in analytical laboratories.

STEMart offers complete instruments and equipments used in GST Pull-Down to facilitate your research. If you want to learn more detail about the related devices, or would like to consult with the experts at STEMart, please feel free to contact us.