GST-pulldown procedure
Summary
The use of recombinant technology to fuse a probe protein with GST (Glutathione S transferase), and affinity binding of the fusion protein to GTH (Glutathione) solidified on a carrier via GST, has been widely used in molecular biology (1) to confirm that two proteins may interact (2) to search for unknown proteins that can interact with known proteins Proteins.
Operation method
GST-pulldown operation procedure
Principle
The probe protein is fused with GST (Glutathione S transferase) using recombinant technology, and the fusion protein binds affinely to GTH (Glutathione) solidified on the carrier via GST. Therefore, proteins interacting with the fusion protein can be adsorbed and separated when they pass through the chromatography column or when they are mixed with the solid-phase complex.GST-pulldown consists of the following three main parts: ① construction of prokaryotic expression vectors with GST tags by recombinant technology; ② expression of fusion proteins with GST tags by prokaryotic expression system; ③ protein purification by GST affinity column to obtain high-purity protein purification. protein purification to obtain high-purity fusion proteins, and then use the GST affinity purification column for protein-protein interaction detection.
Materials and Instruments
Probe Proteins Prokaryotic Proteins Cellular Proteins Tissue Protein Extracts
NaCl KCl Na2HPO4 KH2PO4 Triton-100 IPTG PMSF (phenylmethylsulfonyl fluoride) Cocktaier Immobilized Glutathione Anhydrous Ethanol
Volumetric flasks Cone flasks High-speed centrifuges Cryogenic refrigerators Ultrasonographs EP tubes Chromatography cabinets
Move
Cellular protein lysate, eluent:
PBS and PBS+1% Triton-100
PBS (1L)
NaCl: 8g
KCl: 0.2g
Na2HPO4: 1.44g
KH2PO4: 0.24g
Add 800ml of distilled water, adjust the pH of the solution to 7.4 with HCl, finally add distilled water to 1L, autoclave, store at 4℃.
PMSF (phenylmethylsulfonyl fluoride) MW:174.19 working concentration of 0.1-1mM, here we use 1mM, storage concentration of 100mM, 0.174g PMSF dissolved in 10ml of anhydrous ethanol can be mixed. Keep at -20°C or 4°C.
(The following procedure is for the study of known prokaryotic protein A and eukaryotic overexpression protein B interactions only)
1. Acquisition of prokaryotic fusion protein A
(1) Transfer the recombinant plasmid encoding protein A with GST to BL21 (DE3) strain by plasmidization
(2) Pick a single clone into a 10 ml tube containing 5 ml LB (+100ug/ml Amp) and incubate at 37°C overnight
(3) Transfer the culture to a 1L conical flask containing 500ml LB (+100ug/mlAmp), incubate at 37℃, 225rpm until the OD600≈1.0-1.5, add the appropriate concentration of IPTG, incubate at the appropriate temperature for an appropriate period of time (the conditions of induction need to be adjusted according to the different proteins), 6000g for 10 minutes, centrifugation at 4℃. Collect the bacteria, remove the supernatant, place the bacteria at -20℃ for O/N.
(4) Freeze and thaw the bacteria at room temperature, put them on ice immediately, add 10-20 ml of bacterial lysate (PBS+1% Triton-100+PMSF) to every 500 ml of culture medium, blow and mix well.
(5) Ultrasonic crushing on ice, 2 seconds on, 9 seconds off, 40-60 minutes total. Until lysate is sufficiently clear
(6) 11000rpm, 15 minutes, centrifugation at 4 ℃ to separate the supernatant, -80 ℃ to save for backup
2、Acquisition of eukaryotic fusion protein B: Clone the base sequence encoding the B protein into the eukaryotic expression vector encoding the labeled protein (e.g. HA, or myc), and transfect the cells.
3、After 48 hours, take an appropriate amount of fusion protein GST-A to freeze and thaw on ice
4, Take 50-70ul GST-Beads (Immobilized Glutathione) into EP tubes, rinse once with 800ul PBS+1% Triton-100, mix the freeze-thawed fusion protein GST-A with it, and rotate the binding for 1 hour at 4℃ in the chromatography cabinet.
5, PBS+1% Triton-100 washed 3 times and PBS washed 3 times. Keep 20ul (PBS+Beads) as Offer.
6, At the same time, lysed eukaryotic fusion protein B, depleted the medium, washed once with PBS (RT), added 300ul of lysate (PBS+1% Triton-100+ Cocktaier in the case of 6well), and placed at 4℃ for 30 minutes.
7, Blow the collection to 1.5ml EP tube, ultrasonic crushing. 13000rpm, 15 minutes, centrifugation at 4℃ to take the supernatant.
8, BCA protein quantification (optional) leave 20ul for Offer, the rest of the sample added to the EP tube that has been purified protein, with PBS to make up the liquid to about 600ul, so that the protein can be fully bound to each other! 4 ℃ rotary binding O/N9: PBS + 1% Triton-100 washed 3 times, PBS washed 3 times.
9, 40ul 5×loading buffer to dissolve the protein on the beads, boil for 3 minutes, centrifuge at high speed, Run -SDS PAGE for Western Blot detection. Use HA or myc Blot.
Caveat
The interference of endogenous proteins makes the experiment show more false positive results：
（1） High purity GST fusion proteins can reduce false positives of experiments. Since high purity fusion proteins can reduce false-positive results, obtaining high purity fusion proteins plays an important role in analyzing the results of GST-pull down experiments. At the same time, in order to maximize the biological activity of fusion proteins, soluble fusion proteins are generally preferred when obtaining fusion proteins, so it is crucial to obtain high-purity soluble proteins. The conditions for obtaining soluble proteins mainly include ① the selection of carriers. (2) The selection of soluble protein expression conditions, (3) induction temperature, (4) induction time, (5) the concentration of the inducer, which can not be metabolized by the cell and has a stable concentration.(2) Although GST tags do not affect the folding and function of downstream proteins and promote soluble expression of recombinant proteins, GST tags may affect the correct folding of proteins, so quality control of fusion proteins will make the experimental results more reliable.(3) In experiments, nuclease is sometimes added to eliminate DNA and RNA that may be bridged to proteins, which may also lead to protein-protein interactions.
Common Problems
1. There are two main types of pull down methods in common use: one is performed with an affinity purification column with a histidine tag, and the other is performed with an affinity purification column with a glutathione luciferase tag.
2. The GST-pull down technique is mainly used to verify whether there is interaction between two proteins in vitro, and its use is mainly in two aspects: one is to prove the possible interaction between two known proteins, and the other is used to search for unknown proteins that can interact with known proteins.
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