The principle of GST-Pull Down
? GST Pull-Down experiment is based on GST(Glutathione-S-transferase), that is, glutathione -S- transferase protein, which can be combined with glutathione. GSH is immobilized on agarose beads to form GSH- agarose beads, and the known protein X is expressed by fusion with GST. The obtained GST-X can be combined with GSH- agarose beads. If there is protein Y interacting with protein X in the environment, a "agarose bead -GSH-GST-X-Y" complex will be formed, and the protein interacting with protein X can be separated and detected.
GST Pull-Down experimental method
(Based on the experimental case of Zhong Ding, verify the predicted interaction between two proteins)
Experimental design
Table 1 Pull Down experimental system
No. 1 2 3 4
A-GST+--+
B-His-+-+
GST--+-
Experimental design
1.IPTG induced the expression of fusion proteins of pET28a-B-His and pGEX-4T- 1-A vectors.
1. 1 expression vector was transformed into Escherichia coli Rosseta;
1.2 Select the monoclonal antibody on the transformation plate and inoculate it into a test tube containing 50 μg/mL Amp and shake it overnight at 37℃ to prepare a seed solution;
1.3 The next day, according to 1: 100, it was inoculated in 30 mL LB culture medium with 50 μg/mL Amp, and shaken at 37℃ until the OD600 of the bacteria was 0.8;
1.4 IPTG was added to the bacterial liquid until the final concentration was 0.5 mM, 1 1~28℃ for shaking table culture, and the expression of fusion protein was induced for 4 ~ 8 hours;
1.5 centrifuge the bacterial liquid to discard the supernatant, resuspend the bacterial precipitate with PBS, break it by ultrasonic wave, and centrifuge to get the supernatant;
1.6 Absorb a small amount of supernatant for SDS-PAGE to detect the expression of target protein.
2. Cell fragmentation and protein purification
The purified target protein was obtained by ultrasonic crushing, GST column affinity purification and Ni column affinity purification respectively.
2. 1 GST column purification
(1) Using the low-pressure chromatography system, the cell lysis supernatant was loaded into GST affinity chromatography column with GST Binding-Buffer pre-balance;
(2) washing the chromatographic column with GST Binding-Buffer until the OD280 value of the effluent reaches the baseline;
(3) eluting the target protein with GST Elution-Buffer, and collecting the effluent;
(4) adding the collected protein solution into a dialysis bag, and dialyzing overnight with Tris-HCl;
(5) SDS-PAGE analysis and WB analysis.
2.2 Ni column purification
(1) The supernatant of cell lysis was loaded on Ni-IDA -Sepharose Cl-6B affinity chromatography column with Ni-IDA Binding-Buffer pre-balance by low pressure chromatography system;
(2) washing the chromatographic column with Ni-IDA Binding-Buffer until the OD280 value of the effluent reaches the baseline;
(3) washing the chromatographic column with Ni-IDA Washing-Buffer until the OD280 value of the effluent reaches the baseline;
(4) eluting the target protein with Ni-IDA ELution-Buffer, and collecting the effluent;
(5) adding the collected protein solution into a dialysis bag, and dialyzing overnight with Tris-HCl;
(6) SDS-PAGE analysis and WB analysis.
3.GST-Pull Down experiment
Gst protein interaction pull-down kit (purchased from Thermo company) was used.
3. 1 balanced resin (GST)
(1) Mix the Immobilized Glutathione evenly, and completely resuspend the resin;
(2) each take 100? L 50% resin suspension was put into 4 centrifuge tubes, centrifuged at 1250 ×g for 2 min, and the supernatant was discarded;
(3) use 500? L PBS resuspended the resin, 1250 ×g centrifuged for 2 min to remove the supernatant, and repeated for 3 times.
3.2 Protein Interaction
(1) respectively add A-GST protein and B-His protein into glutathione resin according to table 1, and rotate and mix the mixture at 4 C for overnight incubation;
(2) 1250 ×g centrifuge for 5 min, then discard the supernatant (the supernatant should be cleaned);
(3) use 500? L PBS heavy suspension resin. 1250×g centrifuge for 2 min and discard the supernatant. Repeat 3 times.
3.3 Elution of Bait Protein and Target Protein Complex
(1) Add 50? L Elution Buffer;
(2) Rotate and mix at 2)4°C and incubate for 20 min;
(3) 1250 ×g centrifuge for 2 min to discard the supernatant, and put the centrifuged liquid on ice;
(4) Take 20? L samples were detected by SDS-PAGE and WB.
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