This post describes several methods for extracting bacterial total protein and membrane proteins.

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Bacterial Total Protein and Membrane Protein
Extraction
—Creative BioMart

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This post describes several methods for extracting bacterial total protein and
membrane proteins.

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1. Reagents
Lysate (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 0.5%
Triton X-100, and adjust pH to 8.5-9.0;
Lysozyme; PMSF protease inhibitor; SDS (1%); Trizol lysis solution;
anhydrous ethanol; isopropanol; 0.3 M guanidine hydrochloride;
Hydrophobic protein extract (non-lysing solution): 1% Triton X-114, 150 mM
NaCl, 10 mM Tris-HCl, 1 mM EDTA, and pH adjusted to 8.0.

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2. Equipment
High-speed centrifuge; ultrasound system; dialysis bags; EP tube;
vortex; water bath and so on.

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3. Procedure
1. Extraction of total protein from fresh samples (simple method)
1) Self-made lysis solution (pH 8.5-9.0): 50 mM Tris-HCl, 2 mM EDTA, 100
mM NaCl, 0.5% Triton X-100, pH adjusted to 8.5-9.0; Add 100 μg / ml
lysozyme, 1 μl / ml protease inhibitor PMSF before use.
2) 40 ml of the bacterial solution was at 4 ℃ for 15 minutes. The precipitate
was washed twice with PBS and precipitated by adding 1 ml of the lysate to
suspend the cells.

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3) Ultrasonic crushing, using 300w, 10s ultrasound / 10s interval,
ultrasound 20min, repeated freezing and thawing ultrasound 3 times
to clear the liquid or discoloration.
4) 1000g centrifugal to remove large debris, the supernatant can be
directly denatured after PAGE electrophoresis, or use 1% SDS
solution after dialysis to freeze.
Disadvantages: Western blotting results show that hydrophobic
transmembrane protein extraction efficiency is limited.

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Isolate the total protein from Trizol lysate
1) After Trizol dissolved sample broken, add chloroform layer, 10000g centrifugation
15min under 2-8 ℃, the upper aqueous phase for RNA extraction, and the volume is
about 60% of the total volume.
2) precipitate the DNA in the intermediate layer and the organic phase with ethanol.
Add 0.3ml of anhydrous ethanol each using 1ml Trizol; room temperature for 3min, 2-8
℃ not more than 2000g centrifugation for 5min.
3) Move the supernatant to a new EP tube and precipitate the protein with isopropanol.
Add 1 ml of Trizol to 1.5 ml of isopropyl alcohol at room temperature for 10 min, 12,000
g at 2-8 ℃ for 10 min, and discard the supernatant.

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4) Wash with 95% ethanol containing 0.3 M guanidine hydrochloride.
1ml Trizol add 2ml lotion, room temperature for 20min, 2-8 ℃ 7500g
centrifugation 5min, discard the supernatant; wash 2 times. Finally
add 2ml of anhydrous ethanol, after vortexing, room temperature for
20min, 2-8 ℃ 7500g centrifugation for 5min, discard the supernatant.
5) freeze-dried 5-10min, dissolve in1% SDS solution, repeated blowing;
50 ℃ temperature bath to completely dissolve, 2-8 ℃ 10000g
centrifugation 10min to remove insoluble.

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Extraction of hydrophobic membrane proteins
from fresh samples (Triton X-114 detergent
method)
1) Prepare a hydrophobic protein extract (non-lysate): 1% Triton X-114, 150
mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH adjusted to 8.0.
2) Bacteria were collected at 4 ℃ after 15000g centrifugationl for 15 min. The
cells were washed for 3 times with 1 ml of PBS containing 5 mM MgCl2 and
finally 15000g centrifugation at 4 ℃ for 15 min.
3) Cell precipitation by adding 1ml cold extract, placed at 4 ℃ for 2h, 17000g
centrifugation 10min, remove the precipitate and take the supernatant.
4) The Triton X-114 content in the supernatant was increased to 2%, and 20
mM CaCl2 was added to inhibit partial protease activity and allowed to stand at
37 ℃ for 10 min. The liquid phase and the decontamination phase were fully
separated by centrifugation at 1000 g for 10 min.

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5) The liquid phase and the decontamination phase were separated,
respectively, with 10 times the volume of cold acetone to precipitate on ice
for 45min.
6) Centrifuged at 17000 g for 30 min at 4 ℃ and washed three times with
deionized water.
7) The precipitate was dissolved in 1% SDS solution; determine the protein
concentration, and compare the extraction efficiency of the protein in the
liquid phase and the decontamination phase. Generally, the hydrophobic
membrane protein in the decontamination phase was more suitable for
further protein experiment.
8) SDS-PAGE was used for further analysis of liquid and decontamination
phase of the protein map.

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