GST Pull-Down Protocol
Licia Miller Product Manager
In cells, proteins participate in extensive protein-protein interaction networks that appear as dynamic "protein machines" that assemble and disassemble in response to a constantly changing influx of intracellular, intercellular, and extracellular cues. An initial step in understanding protein structure and function is to determine which proteins interact with each other and thus the relevant biological pathways. Pull-Down technology has become an invaluable tool for life scientists interested in studying cellular pathways through protein-protein interactions .
The Pull-Down technique is an in vitro method for determining the physical interactions between two or more proteins. Pull-Down assays can be used both to confirm the existence of protein-protein interactions predicted by other research techniques (e.g., co-immunoprecipitation) and as a preliminary screening assay to identify previously unknown protein-protein interactions.
Pull-Down technology is a form of affinity purification that is similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. Affinity chromatography (i.e., affinity purification) greatly increases the speed and efficiency of protein purification while providing a technical platform for pull-down or co-purification of potential binding partners.
In the Pull-Down technique, the bait protein is labeled and captured on an immobilized affinity ligand, thereby generating a "secondary affinity support" to purify other proteins that interact with the bait protein. The secondary affinity support with immobilized bait is then incubated with a protein source (such as cell lysate) containing the putative "prey" protein. The source of the prey protein in this step depends on the researcher 's goal for this experiment, whether to confirm a previously suspected protein-protein interaction or to identify an unknown interaction. Following the elution of the protein-protein interaction complex, the complex is analyzed by techniques such as SDS-PAGE. The method of protein elution depends on the affinity ligand and ranges from the use of a competitive analyte to a low pH or reducing buffer.
Among them, GST Pull-Down (GST fusion protein sedimentation technology) is a common method for in vitro detection of protein interactions. A set of control experiments set up to exclude false positives is to bind the sample to GST in the absence of bait protein. The control can be the lysate of transformed cells expressing GST (non-bait fusion protein) or the lysate of non-transformed cells with GST added.
In addition to GST, there are many similar fusion proteins. For example, the "bait" protein fused with Staphylococcus protein A can be purified through a chromatographic column fixed with IgG; the "bait" protein fused with an oligohistidine peptide can be purified through a chromatographic column bound to Ni2+ ; the "bait" protein fused with dihydrofolate reductase can be purified through a chromatographic column fixed with methotrexate, and so on.
The following is a reference experimental process for the GST Pull-Down experiment , which may vary slightly from laboratory to laboratory:
Experimental procedures
1. Construct a prokaryotic or eukaryotic expression vector of bait protein containing GST tag to express fusion protein.
(1) The culture medium with successfully constructed expression vector was transferred to to a 500 ml LB (containing 500 ul ampicillin) in a 1L conical flask, 37°C, 200 rpm for several hours.
(2) Determine the OD600 value. When the OD600 reaches about 0.6, add an appropriate concentration of IPTG and incubate at 20°C and 200 rpm conditions for 10-16 h (preliminary experiments are usually required to determine the optimal IPTG concentration, induction temperature and time) .
(3) After the induction time is appropriate, pour the bacterial solution into 50 ml centrifuge tube, centrifuge 4000 rpm at 4℃ for 10 minutes, discard the supernatant, and collect the bacteria at the bottom of the tube ( if not used temporarily, the bacteria can be stored in a -80℃ refrigerator ).
(4) First add a small amount of PBS into the centrifuge tube and centrifuge for 5-10 min, and discard the supernatant. Then add the corresponding PBS at the rate of 1 ml PBS per 10 ml bacterial solution precipitation and vortexed to fully dissolve the bacterial precipitate in the PBS solution.
(5) Place the mixed cells in an ice water bath and use an ultrasonic device to disrupt them. Ultrasonic disruption should be performed for 20 seconds each time, interval 30 seconds (the crushing time, crushing times and interval time depend on the specific situation). After an appropriate period of ultrasound, the solution will appear clear.
(6) Place the ultrasonic solution at 4°C, 4000 rpm centrifuge for 10 minutes. The supernatant is transferred to a new centrifuge tube and stored at -80 °C for later use.
2. Purification of GST- "bait" fusion protein.
(1) Add an appropriate volume of 50% Glutathione Sepharose 4B to the freshly prepared lysate supernatant and shake slowly on a shaker at 4°C for 30-60 min;
(2) 4°C, Centrifuge at 4000 rpm for 5 min, discard the supernatant, at which point the GST- “bait” fusion protein is bound to the Glutathione Sepharose 4B;
(3) Add 200 ul pre-cooled PBS solution, gently shake the suspended microspheres, wash the Glutathione Sepharose 4B once, 4°C, Centrifuge at 4000 rpm for 5 min, discard the supernatant, and repeat this step 3 times;
(4) Use a pipette to aspirate the liquid on the surface of the microspheres for the last time, but be careful not to aspirate the microspheres, and you will get GST- "bait" fusion protein bound to the Glutathione Sepharose 4B.
3. Preparation of “prey” protein
"Prey" protein can be fused with a His tag for prokaryotic expression, or it can be fused with a Flag/HA/Myc tag for eukaryotic expression.
4. In vitro protein binding
(1) Suspend Glutathione Sepharose 4B bounded with GST- “bait” fusion protein in an appropriate volume of buffer and add 20 ul of solution containing the "prey" protein, and Glutathione Sepharose 4B conjugated with GST protein as a negative control, shake on a shaker for 4-8 h (4℃);
(2) 4°C, 4000 rpm centrifuge for 5 min, discard the supernatant;
(3) Add 200 ul pre-cooled buffer along the wall to wash the Glutathione Sepharose 4B.
(4) 4°C, 4000 rpm centrifuge for 5 min, discard the supernatant, and repeat this step 3 times;
(5) After absorbing the liquid on the Glutathione Sepharose 4B, add 20 ul 1× protein electrophoresis loading buffer, then boiling water bath for 5 min, put into -20℃ refrigerator for later use;
(6) Detection by SDS-PAGE and Western blot.
For more product details, please visit Aladdin Scientific website.