GST pull-down analysis
Summary
Protein-protein interactions play an important role in a variety of life processes such as biocatalysis, transporter, signaling, immunity, cellular regulation, etc. Therefore, the study of such interactions has a profound significance in understanding the laws of life activities.GST pull-down analysis is mainly used in the study of protein-protein interactions.
Principle
The principle of the GST pull-down technique is a method of purifying interacting proteins from non-interacting protein solutions by utilizing the affinity of GST for glutathione-coupled globule beads. It typically has two applications: to identify novel interactions between GST-fused trolling proteins and unknown proteins, and to confirm possible interactions between trolling proteins and known proteins.
The former requires techniques such as direct SDS-PAGE (high abundance target proteins) or isotope-labeled SDS-PAGE (low abundance target proteins) followed by coupled mass spectrometry to determine the "identity" of the target proteins, while the latter can be detected by Western blotting with antibodies against known target proteins.
Appliance
GST pull-down analysis is mainly used for protein-protein interaction studies.
Operation method
GST pull-down analysis
Principle
The principle of the GST pull-down technique is a method of purifying interacting proteins from non-interacting protein solutions by utilizing the affinity of GST for glutathione-coupled globule beads. It typically has two applications: to identify novel interactions between GST-fused trolling proteins and unknown proteins, and to confirm possible interactions between trolling proteins and known proteins. The former requires techniques such as direct SDS-PAGE (high abundance target proteins) or isotope-labeled SDS-PAGE (low abundance target proteins) followed by coupled mass spectrometry to determine the "identity" of the target proteins, while the latter can be detected by Western blotting with antibodies against known target proteins.
Materials and Instruments
Equipment:
① Equipment for cell passaging culture
② 3.5 cm diameter plastic petri dishes (or 50 ml culture flasks), ③ 4 ℃ chromatography cabinets or cold rooms, ③ 4 ℃ chromatography cabinets or cold rooms.
③ 4 ℃ chromatography cabinet or cold room.
③ 4 ℃ chromatography cabinet or cold room, ④ Flip sample rotator, boiling water bath, ⑤ Electrophoresis apparatus, vertical plate
⑤ Electrophoresis apparatus, vertical plate electrophoresis tanks
⑤ Electrophoresis apparatus, vertical plate electrophoresis tank ⑥ Radiographic autoradiography film cassette
⑦ Equipment for SDS-PAGE and Western blotting.
Reagents:
① Material: Cells to be used for the study
② Appropriate cell culture medium
③ Pre-purified GST fusion bait protein
④ Glutathione-agarose beads
⑤ Antibody against catch protein (monoclonal or polyclonal antibody, for Western blotting)
⑥ Salts such as NaCl, MgCl2, CaCl2, KCI, Na2HPO4 - 12H2O, KH2PO4, HEPES, and so on.
(vii) Triton X- 100, mercaptoethanol, etc.
(8) Reagents for SDS-PAGE and Western blotting.
Move
The basic process of GST pull-down analysis can be divided into the following steps: (i) Reagent preparation
(1) Lysis buffer 1% Triton X-100; 150 mmol/L NaCl; 2 mmol/L MgCl2; 2 mmol/L CaCl2; 10 mmol/L HEPES, pH 7.4. Add protease inhibitors [1 μg/ml Leupepin, 1 μg/ml Antipain, 1 μg/ml Phenyl-methylsulphuryl fluoride, 1 μg/ml Phenylmethylsulphuryl fluoride, 1 μg/ml Phenylmethylsulphuryl fluoride, 1 μg/ml Phenylmethylsulphuryl fluoride 1 μg/ml Leupepin, 1 μg/ml Antipain, 1 μg/ml Phenyl-methylsulphonylfluoride (PMSF), 1.25 μg/ml Pepstatin].
(2) PBS buffer NaCl, 8.0 g; KC1, 0.2 g; Na2HPO4 - 12H2O, 2.9 g; KH2PO4, 0.2 g; double-distilled water to 1000 ml. autoclaved (usually at 1.034x105 Pa for 30 min).
(3) Wash buffer 0.1% Triton X-100; 10 mmol/L mercaptoethanol; PBS was prepared.
(ii) Cell lysis(1) Culture the cells in culture flasks to 80% confluence.
(2) Collect the cells by centrifugation at 1000 r/min for 5 min and wash twice with 10 ml PBS.
(3) Add 1 ml of lysis buffer to the cell sediment and place on ice for 20 min.
(4) Centrifuge at 12000 g for 10 min at 4 ℃ and transfer the supernatant into a new Eppendorf tube.
(iii) Binding of GST fusion bait protein (1) Dilute 100 μL of cell lysate with 900 μL of lysis buffer, and add 10 μg of GST fusion trolling protein.(2) Shake at 4 ℃ overnight.
(3) Add 80 μL of glutathione-agarose spheres (50% suspension) and shake at 4 ℃ for 4 hours.
(4) Centrifuge at 750 g for 5 min at 4 ℃.
(5) Wash the spheres with 1 ml of washing buffer and centrifuge at 750 g for 5 min at 4 ℃. repeat the washing twice.
(iv) Elution(1) Resuspend the precipitated spheres with 30 μL of 2xSDS sample buffer.
(2) Heat at 95 ℃ for 5 min.
(3) Centrifuge the supernatant, which was subsequently used for SDS-PAGE and Western blot analysis.
(E) SDS-PAGE and Western blotting(1) Equilibrate the gel in transfer buffer for 10 min. Note: This step can be omitted if small molecule proteins are detected, as they tend to diffuse out of the gel.
(2) Cut 6 pieces of membrane and filter paper according to the size of the gel, and equilibrate them in the transfer buffer for 10 min. If PVDF membrane is used, it should be saturated with pure methanol for 3-5 seconds.
(3) Assemble the transfer sandwich: sponge 3 layers of filter paper adhesive membrane 3 layers of filter paper sponge, after each layer is put in place, use a test tube to drive away the air bubbles. Remember: the adhesive is placed on the negative side (black side).
(4) Place the transfer bath in an ice bath, put in the sandwich (black side on black side), add transfer buffer, insert the electrode, 100 V, 1 h (current about 0.3 A). Note: The sandwich and electrodes should be checked again for correct assembly and that the power supply is on.
(5) Wash the membrane with 25 ml TBS for 5 min at room temperature with shaking.
(6) Place the membrane in 25 ml blocking buffer for 1 h, room temperature, shaking. 15 ml TBS/T wash 3 times (5 min/T).
(7) Add primary antibody at appropriate dilution and incubate for 1-2 h at room temperature or overnight at 4 °C with slow shaking. 15 ml TBS/T washes 3 times (5 min/T).
(8) Add the appropriate dilution of alkaline phosphatase (AP) or horseradish peroxidase (HRP)-labeled secondary antibody and incubate at room temperature for 1 h with slow shaking. 15 ml TBS/T washes 3 times (5 min/T). 15 ml TBS washes once.
(9) Protein assay (colorimetric or luminescent method, according to the instructions of the corresponding reagents).
(F) Experimental results and analysisDetermine whether there is interaction between the proteins by whether the bands are visible or not.
Caveat
(1) Conditions need to be optimized for each protein for GST pull-down experiments.① Composition of the buffer in which the interaction occurs Many buffers are available, but the efficiency of the interaction may be affected by the buffer used.② Amount of target protein to be mixed with the fusion bait protein The amount of material required is mainly determined by the abundance of the target protein and the affinity of the interaction, which are usually unknown at the beginning of the experiment and need to be mapped out step by step.(③) Conditions for washing the spherical beads Buffers containing different salt and denaturant concentrations are used to eliminate non-specific interactions, so their strength needs to be controlled properly, otherwise inadequate washing or disruption of specific binding may result.(2) Pay attention to the setting of controls, including the total protein added, the pull-down sample of non-fusion GST, etc. If the experiment fails, the problem can be found by analyzing the supernatant collected from the total protein lysate in step 1.(4) and step 2.(4), the eluent of the GST control, the eluent of the spherical beads and the control of the cell lysate as well as the composition of eluted spherical beads by Western blotting. .(3) The protease inhibitors PMSF, mercaptoethanol, and acrylamide are highly toxic and are handled as specified.
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