GST fusion proteins, as recombinant proteins expressed in bacteria, have been used in thousands of research projects since their introduction (Smith and johnson 1988). They are often used to prepare antibodies which can be used to study protein-protein interactions and to analyze biochemical reactions. This experiment is from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Protein-protein interaction assay with GST fusion proteins
Materials and Instruments
GST fusion protein bound to glutathione-globose Move makings For more product details, please visit Aladdin Scientific website.
Alkaline buffer Closed buffer Interaction buffer PK buffer Reduced glutathione in Tris-Cl Wash buffer 1 Wash buffer 2 Protease Protein kinase A [y-32P]ATP
Membrane or Filter Membrane Bound to Protein to be Screened Sephadex G-50 Transfer Columns
Buffers and solutions
Dilute the buffer to the appropriate concentration.
Alkaline buffer
20 mmol/L HEPES (pH 7.5)
50 mmol/LKCl
10 mmol/LMgCl2
1 mmol/L dithiothreitol
0.1%NP-40
Closed buffer
5% skimmed milk powder in alkaline buffer
Interaction Buffer
1% skimmed milk powder in alkaline buffer
2xPK buffer
100 mmol/L KPO4 (as in the original English book - translator's note)
20 mmol/LMgCl2
10 mmol/LNaF
9 mmol/L dithiothreitol
Reduced glutathione (20 mmol/L) in 50 mmol/L Tris-Cl (pH8.0)
Optional, see step 3
Wash Buffer 1
Phosphate buffer salt solution containing 0.2% TritonX-100
Wash Buffer 2
Phosphate buffer solution containing 0.2% TritonX-100 and 100 mmol/LKCl
Enzyme and buffer
Protease
Optional, see step 3.
Protein kinase A
Freshly prepared according to product instructions for each use.
Radioactive Compounds
[ y-32P ]ATP (6000Ci/mmol)
Specialized equipment
Membrane or filter membrane bound to the protein to be screened.
See step 5.
Sephadex G-50 Transfer Columns
Optional, see step 4.
Additional Reagents
Step 3 of this protocol requires a number of reagents that cleave the fusion protein from its carrier or from bound glutathione-agarose, see Chapter 15, Protocol 5.
Step 5 of this protocol requires polyacrylamide gels containing the proteins to be detected (see Appendix 8) or plates containing cDNA expression libraries (see Chapter 14, Scheme 2), as well as immunoblotting reagents as described in Appendix 8.
GST alone or a negative control for a non-specific protein should be added to the SDS polyacrylamide gel together with the target protein. If the target protein is a member of a conserved family, the interactions of the target protein can be compared with other proteins of the same family.
Vectors and Strains
GST fusion proteins bound to glutathione-globose
This protocol is suitable for fusion proteins containing protein kinase phosphorylation sites (Ron and Dressler 1992). Vectors containing cAMP-dependent protein kinase recognition sequences can also be used (Amersham Pharmacia Biotech). If untagged GST fusion proteins are used, see the text box at the end of this protocol, "Alternative: detection of protein-protein interactions with anti-GST antibodies."
Methods
Preparation of radiolabeled protein probes
1. Prepare the following reaction mixture in a microcentrifuge tube:
[ γ-32P ]ATP (6000Ci/mmol) 5ul
Protein kinase A 1unit/ul
GST fusion protein on glutathione agarose 1~3ug
2XPK buffer 12.5ul
Water to 25ul
Incubate the reaction mixture at 37°C for 30 min.
The fusion protein may be cleaved by protease prior to the labeling reaction. In this case, replace the protein bound to glutathione agarose in the labeling reaction with the cleaved protein and incubate at 37°C for 30 min, then proceed to step 4.
The GST component is retained on the fusion protein, and the experiment is repeated using the GST itself (bound to glutathione agarose) as a control. This may not be practical in library screening, so it is important to test the ability of the positive clone to bind to GST itself. This control is usually done after the fourth purification and before clone identification.
2. After the labeling reaction is complete, the agarose beads are washed by adding 200ul of 1XPK buffer to a centrifuge tube and then centrifuged in a microcentrifuge at maximum speed for 1 min. The supernatant containing the free radionuclides is discarded in the appropriate manner and the wash is repeated.
3. Wash the labeled GST fusion proteins from the agarose beads by cutting down the labeled proteins with a protease or by washing the labeled GST fusion proteins with 20 mmol/L reduced glutathione in 50 mmol/L Tris (pH 8.0) (see Chapter 15, Scheme 5). Store the labeled proteins in an ice box and use them on the same day.
4. If the labeled protein is separated from the GST component prior to the labeling reaction (see Note for Step 1), place the labeled protein on a SephadexG-50 column equilibrated with 1XPK buffer to remove free labeled nucleic acid. Once the probe protein is separated from the free nucleic acid, it is ready for use. Store the labeled proteins in an ice box and use them on the same day.
Probe Membrane
5. Prepare the membrane to be probed by transferring the protein to the membrane using standard techniques.
Proteins transferred from SDS-polyacrylamide gels can usually be detected directly. If proteins are transferred from a cDNA expression library, an additional protocol, "Refolding of Membrane Bound Proteins", may be performed before proceeding to step 6.
6. Completely cover the membrane with alkaline buffer and gently shake at 4°C for 10 minutes.
7. Discard the basic buffer, completely cover the membrane with the blocking buffer and incubate at 4°C with gentle shaking for 4 h to overnight.
8. Add 1 to 3ug of storage probe (from step 3 or 4) to enough interaction buffer to make a final concentration of 1 to 5 nmol/L to prepare a labeled protein solution. Transfer the membrane to a dish containing the diluted probe and make sure that the probe solution is in uniform contact with the entire surface of the membrane. incubate at 4°C with gentle shaking for 4~5 h.
9. Discard the radioactive probe solution in an appropriate manner. Completely cover the membrane with Paint Wash Buffer 1 and incubate for 10 min at 4°C with gentle shaking. repeat the wash three times.
10. Completely cover the membrane with Wash Buffer 2 and wash for 10 min at 4°C with gentle shaking. repeat the wash once.
11. Carefully wrap the membrane in plastic wrap and expose to x-ray film. 
