GST fusion protein sedimentation assay
GST fusion protein sedimentation assay
The GST sedimentation experiment has an advantage over far western (Scheme 2): the probe protein is incubated with a possible partner protein in a more natural environment, thus enhancing the effectiveness of the interaction. This experiment is from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook D.W. Russell.
Operation method
GST Fusion Protein Sedimentation Technology for Protein Detection
Materials and Instruments
Cell lysate in which protein 35S is labeled Move makings For more product details, please visit Aladdin Scientific website.
Lysis buffer SDS Polyacrylamide gel Probe GST Protein
Boiling water bath Turnover omicron spinner Glutathione agarose beads
Buffers and solutions
Dilute the buffer to the appropriate concentration.
Lysis buffer
20 mmol/LTris-Cl (pH 8.0)
200 mml/L NaCl
1 mmol/L EDTA(pH8.0)
0.5% Nonidet P-40
Protease inhibitors were added before use to reach the following final concentrations: 2ug/ul aprotinin, 1ug/ul leupeptin, 0.7ug/ml pepstatin and 25ug/ml phenylmethylsulfonyl fluoride (PMSF). PMSF)
Reduced glutathione (20 mmol/L) dissolved in 50 mmol/L Tris-Cl (pH 8.0).
Optional, see Step 8.
2xSDS-PAGE Sampling Buffer
Gel
SDS polyacrylamide gel
See Appendix 8 for details on SDS polyacrylamide gel electrophoresis.
Specialized Equipment
Boiling water bath
Turnover auspicious product rotator
Glutathione Agarose Beads
Make a 50% suspension in lysis buffer.
Probe
GST Protein
GST fusion proteins with "bait" or probe sequences
GST fusion proteins with "bait" or probe sequences are constructed as described in Scheme 1 in Chapter 15.
Cells and Tissues
Cell lysates, in which protein 35S is labeled with the
Depending on the purpose of the experiment and the desired assay, unlabeled cell lysates may be used. For further details, please see the protocol description.
Additional reagents
Step 11 of this protocol requires immunoblotting and staining reagents as listed in Appendix 8 for proteins that are separated by SDS polyacrylamide gel electrophoresis.
Methods
Pre-cleared cell lysate
1. Incubate the cell lysate with 50ul of 50% glutathione agarose bead suspension and 25ug GST at 4°C for 2 h. The amount of lysate required to detect interactions is highly variable. Start with a lysate equivalent to 1X106-1X107 cells.
Since the purpose of the experiment is to compare GSTT to GST fusion proteins, it is necessary to prepare a sufficient amount of pre-cleared lysate for each reaction. Effective mixing of the reagents is essential for success. For this purpose, the reaction should be carried out in a reasonable volume: a good starting value is 500~1000ul.
This pre-cleaning step is to remove from the lysate proteins that have non-specific interactions with the GST component or beads. Pre-cleaning with GST or glutathione agarose beads is not always necessary if the interaction is to be detected primarily with antibodies against candidate interacting proteins. However, these steps help to reduce the background when cell lysates labeled with 35S are used to identify novel protein-protein interactions. If interactions are being detected with antibodies against candidate interacting proteins, it is important to include two controls: GST plus spheroplasts and spheroplasts only.
If the interacting target protein is known to be present at a specialized cell site. Then the probe should be incubated with a cell lysate fraction corresponding to that cell site.
2. Centrifuge the mixture in a microcentrifuge at 4°C for 2 min at maximum speed.
3. Transfer the supernatant (i.e. the pre-cleared cell lysate) to a new centrifuge tube.
Detection of cell lysate
4. Set up two microcentrifuge tubes containing equal amounts of pre-cleared cell lysate and 50ul of glutathione agarose beads. Add approximately 10ug of GST protein to one tube and approximately 10ug of GST fusion probe protein to the other tube. The amount of probe and control proteins added to both reactions should be equimolar (i.e., the final concentration of GST should be the same as the GST fusion probe protein). Incubate the mixed mixture in centrifuge tubes for 2 h at 4°C with the tubes turned over.
Important: If bound proteins are removed from the beads by boiling (step 10), it is important to introduce a control containing only glutathione agarose beads and cell lysate to detect proteins that bind non-specifically to the beads.
5. Centrifuge the samples on a microcentrifuge for 2 min at maximum speed.
6. Collect the supernatant in a new microcentrifuge tube. These samples can be analyzed by SDS polyacrylamide gel electrophoresis in step 10.
7. Wash the spherical beads with 1 ml of ice-cold lysate. Centrifuge in a microcentrifuge at maximum speed for 1 min. discard the supernatant. Repeat three times.
8. (Optional) Add 50ul of 20 mmol/L reduced glutathione (in 50 mmol/L Tris-Cl pH 8.0) to the spherical beads to elute the GST fusion protein and any bound proteins. Centrifuge on a microcentrifuge for 2 min at maximum speed.
9. Mix the beads (from step 7) or eluted proteins (from step 8) with an equal volume of 2XSDS-PAGE Sampling Buffer.
Detection of interacting proteins
10. Boil the sample for 4 min and analyze by SDS polyacrylamide gel electrophoresis.
11. The method for detecting proteins that bind to GST fusion proteins depends on whether the cell lysate is 35S-labeled or not and the purpose of the experiment.
(1) If the goal is to detect all 35S-labeled proteins bound to the fusion protein, the gel is dried on a gel dryer and exposed to X-ray film for radioautography.
(2) If the purpose was to detect specifically bound proteins, the proteins were transferred from the SDS polyacrylamide gel to the membrane for immunoblot analysis (Appendix 8).
(3) If the objective is to determine the size and abundance of proteins bound to the fusion protein, and these proteins are from non-radioactive lysates, the gel is stained with either Caumas Brilliant Blue or silver nitrate (Appendix 8). 