Bacterial Total Protein and Membrane Protein Extraction Protocol

Main Reagents

Lysis solution (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, adjust pH to 8.5-9.0 and set aside;
Hydrophobic protein extract (not lysate): 1% Triton X-114, 150mM NaCl, 10mM Tris-HCl, 1mM EDTA, adjust pH to 8.0 and reserve;
Lysozyme; protease inhibitor PMSF; 1% SDS solution; Trizol lysate; anhydrous ethanol; isopropanol; 0.3 M guanidine hydrochloride.

Main Equipment

High-speed centrifuge; ultrasound instrument; dialysis bag; EP tube; vortex instrument; water bath, etc.

Experimental Steps

1. Extraction of total protein from fresh samples (simple method)

(1) Lysis solution (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5% Triton X-100, pH 8.5-9.0; add 100 μg/ml lysozyme and 1 μl/ml protease inhibitor PMSF before use. the amount of lysis solution is 10-50 ml lysis solution/1 g wet bacteria.

(2) Centrifuge 40 ml of bacterial solution at 12000 g, 4°C for 15 min to collect the bacteria, the precipitate was washed twice with PBS suspension, the precipitate was added to 1 ml of lysate to suspend the bacteria.

(3) Ultrasonic crushing, using 300 w, 10 s sonication/10 s interval, 20 min sonication, repeated freeze-thaw sonication 3 times until the bacterial solution becomes clear or discolored.

(4) Centrifuge at 1000 g to remove large debris, the supernatant can be denatured directly and detected by PAGE, or lyophilized after dialysis with 1% SDS solution.

2. Total protein isolation from Trizol lysate

(1) Trizol-solubilized samples were ground and crushed, layered with chloroform, and centrifuged at 10,000g for 15 min at 2-8°C. The upper aqueous phase was used for RNA extraction at a volume of about 60% of the total volume.

(2) Precipitate the DNA in the middle layer and organic phase with ethanol. add 0.3 ml of anhydrous ethanol per 1 ml of Trizol used and mix well, leave at room temperature for 3 min and centrifuge at 2-8°C for 5 min at no more than 2000 g.

(3) Transfer the supernatant to a new EP tube and precipitate the protein with isopropanol. Add 1.5 ml of isopropanol for every 1 ml of Trizol used, leave at room temperature for 10 min, centrifuge at 12,000 g for 10 min at 2-8°C, and discard the supernatant.

(4) Wash with 95% ethanol containing 0.3 M guanidine hydrochloride. Add 2 ml of washing solution per 1 ml of Trizol, leave at room temperature for 20 min, centrifuge at 7500g for 5 min at 2-8°C, discard the supernatant, and repeat the washing twice. Finally, add 2 ml of anhydrous ethanol, vortex and leave for 20 min at room temperature, centrifuge at 7500 g for 5 min at 2-8°C, and discard the supernatant.

(5) Freeze dry for 5-10 min, dissolve in 1% SDS solution, blow repeatedly, warm bath at 50°C to dissolve completely, centrifuge at 10,000 g for 10 min at 2-8°C to remove insoluble material.

(6) Alternative solution: The phenol supernatant in (3) was transferred to a small molecular weight dialysis bag and dialyzed 3 times in 1% SDS solution at 2-8°C. The precipitate was removed by centrifugation at 1000g for 10 min and the supernatant could be used directly for protein experiments.

3. Extraction of hydrophobic membrane proteins from fresh samples (Triton X-114 detergent method)

(1) Prepare hydrophobic protein extract (not lysate): 1% Triton X-114, 150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, adjust pH to 8.0 and set aside.

(2) Bacteria were collected by centrifugation at 15,000 g for 15 min at 4°C. Bacteria were washed 3 times with 1ml of PBS containing 5 mM MgCl2 and finally collected by centrifugation at 15,000 g for 15 min at 4°C.

(3) The precipitate of the bacterium was added to 1 ml of cold extract, placed at 4℃ for 2 h, centrifuged at 17000 g for 10 min, and the supernatant was removed from the precipitate.

(4) The Triton X-114 content in the above supernatant was increased to 2%, and then 20 mM CaCl2 was added to inhibit part of the protease activity, and placed at 37°C for 10 min to make the stratification. Centrifuge at 1000 g for 10 min at room temperature to make the liquid phase and the decontaminated phase fully stratified.

(5) The liquid phase and the decontaminated phase were separated and precipitated on ice with 10 times the volume of cold acetone for 45 min.

(6) The precipitate was centrifuged at 17,000 g for 30 min at 4°C and washed three times with deionized water.

(7) The precipitate was dissolved in 1% SDS solution and the protein concentration was measured to compare the protein extraction efficiency in the liquid phase and the decontaminated phase, which is generally more hydrophobic membrane proteins in the decontaminated phase and suitable for further protein experiments.

(8) SDS-PAGE was used to further analyze the protein profiles of the liquid and decontaminated phases.

* For research use only. Not intended for any clinical use.

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Bacterial Total Protein and Membrane Protein Extraction Protocol

Main Reagents

Main Equipment

Experimental Steps

1. Extraction of total protein from fresh samples (simple method)

2. Total protein isolation from Trizol lysate

3. Extraction of hydrophobic membrane proteins from fresh samples (Triton X-114 detergent method)

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