Bacterial total protein and membrane protein extraction method
Experimental reagent
Lysate (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, adjusted to pH 8.5-9.0;
Lysozyme; protease inhibitor PMSF; 1% SDS solution; Trizol lysate; absolute ethanol; isopropanol; 0.3 M guanidine hydrochloride;
Hydrophobic protein extract (non-lysate): 1% Triton X-114, 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, adjusted to pH 8.0.
Laboratory equipment
High-speed centrifuge; ultrasound system; dialysis bag; EP tube; vortex device; water bath.
Experimental procedure
1. Extract total protein from fresh samples (simple method)
1) Self-dispensing lysate (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, adjust the pH to 8.5-9.0, and add 100 μg/ml before use. Lysozyme, 1 μl/ml protease inhibitor PMSF. The lysate is used in an amount of 10 to 50 ml of lysate per 1 g of wet cells.
2) 40 ml of the bacterial solution was centrifuged at 12,000 g for 15 minutes at 4 ° C to collect the cells, and the precipitate was washed twice with PBS, and the precipitate was added to 1 ml of the lysate to suspend the cells.
3) Ultrasonic pulverization, using 300w, 10s ultrasound / 10s interval, ultrasound 20min, repeated freeze-thaw ultrasound 3 times to clear or discoloration of bacterial liquid.
4) 1000g is centrifuged to remove large fragments. The supernatant can be directly denatured and detected by PAGE electrophoresis, or dialyzed with 1% SDS solution and frozen.
Disadvantages: Western blotting results show that the hydrophobic transmembrane protein extraction efficiency is limited.
2. Isolation of total protein from Trizol lysate
1) Trizol dissolved sample was ground and crushed, layered with chloroform, centrifuged at 10,000 g for 15 min at 2-8 ° C, and the upper aqueous phase was used for RNA extraction, and the volume was about 60% of the total volume.
2) Precipitate the DNA in the intermediate layer and the organic phase with ethanol. Each time, 1 ml of Trizol was added to 0.3 ml of absolute ethanol, and the mixture was allowed to stand at room temperature for 3 minutes, and centrifuged at 2-8 ° C for not more than 2000 g for 5 minutes.
3) Transfer the supernatant to a new EP tube and precipitate the protein with isopropanol. 1.5 ml of isopropanol was added per 1 ml of Trizol, left at room temperature for 10 min, centrifuged at 12000 g for 2 min at 2-8 ° C, and the supernatant was discarded.
4) Wash with 95% ethanol containing 0.3 M guanidine hydrochloride. 2 ml of the washing solution was added per 1 ml of Trizol, left at room temperature for 20 min, centrifuged at 7500 g for 2 min at 2-8 ° C, the supernatant was discarded, and the washing was repeated twice. Finally, 2 ml of absolute ethanol was added, vortexed and left at room temperature for 20 min, centrifuged at 7500 g for 2 min at 2-8 ° C, and the supernatant was discarded.
5) Freeze-drying for 5-10 min, 1% SDS solution is dissolved, repeatedly blown, completely dissolved in a 50 °C warm bath, and centrifuged at 10000 g for 10 min at 2-8 ° C to remove insoluble matter.
6) Alternative: Transfer the phenolic alcohol supernatant from 3 to a small molecular weight dialysis bag, dialyze 3 times in 1% SDS solution at 2-8 ° C, centrifuge at 1000 g for 10 min to remove the precipitate, and use the supernatant directly for protein. experiment.
3. Extraction of hydrophobic membrane proteins from fresh samples (Triton X-114 detergent method)
1) Formulation of hydrophobic protein extract (non-lysate): 1% Triton X-114, 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, adjusted to pH 8.0 for use.
2) The bacterial solution was centrifuged at 15000 g for 15 min at 4 ° C to collect the cells; the cells were washed 3 times with 1 ml of PBS containing 5 mM MgCl 2 , and finally the cells were collected by centrifugation at 15000 g for 15 min at 4 ° C.
3) The cell pellet was added with 1 ml of cold extract, placed at 4 ° C for 2 h, centrifuged at 17000 g for 10 min, and the supernatant was removed.
4) The content of Triton X-114 in the above supernatant was increased to 2%, and 20 mM CaCl2 was added to inhibit partial protease activity, and the layer was allowed to stand at 37 ° C for 10 min. The liquid phase and the decontamination phase were sufficiently stratified by centrifugation at 1000 g for 10 min at room temperature.
5) The liquid phase and the decontamination phase were separated and precipitated on ice for 10 min with 10 volumes of cold acetone, respectively.
6) Centrifuge at 17000 g for 30 min at 4 ° C, and wash the pellet 3 times with deionized water.
7) Dissolve the precipitate in 1% SDS solution, determine the protein concentration, compare the protein extraction efficiency in the liquid phase and the decontamination phase, generally the hydrophobic membrane protein in the decontamination phase is more suitable for further protein experiments.
8) Further analysis of the protein profiles of the liquid phase and the decontamination phase by SDS-PAGE.
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