This experiment describes the application of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, also known as GST co-binding purification methods. This co-binding technique can be used to complement other methods for assessing protein-protein interactions: in vitro assays such as electrophoretic mobility shift analysis (EM- SA) or immunoprecipitation; or in vivo assays such as two-hybrid interaction traps and ubiquitin-based sensors for shedding proteins. Source: Compact Laboratory Guide to Molecular Biology, Fifth Edition.
Operation method
GST co-conjugation purification method
Principle
Agarose particles and bait protein complexes are not lost during washing and all reactions contain equal amounts of bait protein. To demonstrate that no material is lost during washing, parallel SDS-PAGE gel electrophoresis should be done using 1/10 of the GST fusions spiked and stained in order to compare the GST fusion protein bands more precisely. In addition, degradation of the fusion protein may result in a reduction in the amount of bait protein, especially if a crude extract (see Fig. 19.2.1) is used instead of purified labeled experimental proteins. The amount of degradation must be accounted for when quantifying the bound experimental protein.
Materials and Instruments
E.coli extract (dissolved in agarose particle binding buffer) [35S] Met-tagged protein GST fusion protein (GST and bound to agarose particles) Move 1. Thaw the E.coli soluble protein extracts on ice. 2. 2. 500 μl of thawed E.coli extract for each reaction is centrifuged for 30 min at 4°C, 175,000 g (70,000 r/min using a Beckman TLA-100 rotor). 3. The supernatant (not less than 400 μl) is transferred to 2 tubes of 200 μl each and placed on ice. 3. Transfer the supernatant (not less than 400 μl) to 2 tubes of 200 μl each and store in an ice bath. 4. 4. Add 1-5 μl of [35S ] methionine-labeled experimental protein to a tube containing 200 μl of the supernatant obtained in step 3 and keep on ice for 15 min. 5. Remove insoluble protein aggregates from the in vitro transcription mixture by microcentrifugation at maximum speed for 15 min at 4 °C. Transfer 200 μl of supernatant (containing labeled experimental proteins and extracts) to a clean test tube. 6. To a separate tube containing 200 μl of the supernatant obtained in step 3, add 20 μl of GST or GST fusion protein (bait protein) bound to agarose particles and mix well. Each reaction requires 2 to 5 μg of GST bait protein fusion. It is preferable to add approximately 20 μl of agarose particles to each reaction so that precipitation of the agarose particles can be seen during washing. However, if 20 μl of agarose pellet binds more than 2-5 μg of protein, the necessary volume can be provided by adding agarose pellet without glutathione. 7. 7. Add 200 μl of the mixed agarose pellet extract suspension (step 6) to the experimental protein-containing extract (step 5). Incubate at 4°C with slow shaking for 1~2 h. 8. Incubate at 4°C at maximum speed. 8. Microcentrifuge the reaction at maximum speed for 1 min at 4°C to precipitate the agarose pellet. The supernatant was removed and the agarose pellet precipitate was washed three times with 1 ml of agarose pellet binding buffer. Each wash is followed by microcentrifugation at maximum speed for 1 min at 4 °C. All liquid should be carefully removed after the last wash. Remove all liquid to ensure an equal amount of sample is added to the analytical SDS-PAGE gel. The last few microliters of liquid can be removed with a thin, twisted strip of filter paper or an SDS spiking pipette with a very thin tip. 9. 9. 25 μl of 1 x SDS Sample Buffer is added directly to each tube and placed in a boiling water bath for 5 min. analytical SDS-PAGE gel electrophoresis is performed. The amount of sample required varies depending on the bait/target protein pair and the assay. Adding all of the sample avoids the problem of sample volume, since there is no uncertainty about the ratio of added to unadded sample. 10. Optional: staining with Caulmers Brilliant Blue. 11. Fix the gel in gel fixative for 30 min at room temperature with slow shaking. Dry the gel and do radioautography or place it on a screen that stores phosphorescent photons. Characterize the radiographs using a scanning optical densitometer or phosphorimager. For more product details, please visit Aladdin Scientific website.
1 × SDS sample buffer Gel fixative (10% glacial acetic acid/45% methanol)
Centrifuge and Beckman TLA-100 rotor Microcentrifuge 4°C X-ray film/storage phosphor screen Scanning optical densitometer/phosphor visualizer