A kind of preparation method of two-dimensional electrophoresis sample of yellow rice wine body protein
A two-dimensional electrophoresis, protein technology, applied in the field of rice wine detection, can solve problems such as affecting protein separation
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Embodiment 1
[0031] The influence of embodiment 1 ammonium acetate-methanol solution on sample quality
[0032] (1) Take a certain volume of rice wine sample, add 2.0g / L calcium chloride, 2.0M sodium hydroxide to adjust the pH to 8.5, and freeze at 4°C overnight.
[0033] (2) Centrifuge at 10,000 g for 30 min at 4° C., and concentrate the obtained supernatant 10 times with a dialysis membrane embedded in PEG20,000 with a molecular weight cut-off of 3,500 Da.
[0034] (3) Desalting the concentrated liquor through a Sephacryl G25 column. Add 5 times the volume of 0.1M ammonium acetate-methanol solution (methanol is the solvent and ammonium acetate is the solute) to the desalted wine sample, and place it in a -20°C refrigerator for precipitation for more than two hours. Centrifuge at 12000g for 30min at 4°C to collect the precipitate. The precipitate was washed twice with -20°C pre-cooled methanol.
[0035] (4) Place the obtained precipitate at room temperature for 10-15 minutes to volatil...
Embodiment 2
[0039] TCA-acetone method:
[0040] (1) Take a certain volume of rice wine sample, add 4 times the volume of 20% TCA acetone solution, and put it in a refrigerator at -20°C overnight.
[0041] (2) Centrifuge at 10000g for 30min at 4°C to obtain the precipitate of rice wine body protein.
[0042] (3) The precipitate was washed twice with propanol pre-cooled at -20°C.
[0043] (4) Place the obtained precipitate at room temperature for 10-15 minutes to volatilize all the methanol. Dissolve the precipitate with hydration solution, control the protein concentration at about 1 mg / mL, and perform two-dimensional electrophoresis.
[0044] The 2D electrophoresis spectrum of rice wine body protein by TCA-acetone method is shown figure 2 .
[0045] From figure 2 It can be seen that under the same electrophoresis conditions as in Example 1, the proteins in the rice wine body prepared in this example piled up after the electrophoresis, and there were no obvious protein spots, so it ...
Embodiment 3
[0046] The influence of embodiment 3 saturated ammonium sulfate precipitation on sample quality
[0047] When using the traditional saturated ammonium sulfate method to precipitate the protein in rice wine body, it was found that only a small amount of protein was precipitated. Two-dimensional electrophoresis and wine body protein composition analysis cannot be performed.
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