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Extraction method of biological membrane proteins and preparation method of electrophoresis sample

An extraction method and protein technology, applied in the preparation of test samples, etc., can solve the problems of stagnation in macroproteomics environment research, difficulty in sample purification, and ineffective results, so as to reduce the interference of electrophoresis, clear protein spots, clear strip effect

Inactive Publication Date: 2015-06-10
TONGJI UNIV
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  • Abstract
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Problems solved by technology

Moreover, the humus produced during the aging process of the biofilm will combine with the proteins released and secreted by the microorganisms, which is difficult to separate and makes the purification of the samples difficult, which is the biggest difficulty in this research.
In addition, protein technology has no amplification method similar to PCR, etc., and it is more difficult to analyze samples with low protein content.
Various existing protein extraction methods are unsatisfactory when applied to biofilms. At present, there is no general and effective method for extracting total proteins from biofilms. This is also the most important reason for the lag in environmental research of metaproteomics.
[0007] The method established by Wilmes.P. is currently widely used by metaproteome researchers. The inventor repeated this method to extract proteins from biofilms, but did not obtain ideal results, indicating that the method lacks good versatility.

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  • Extraction method of biological membrane proteins and preparation method of electrophoresis sample
  • Extraction method of biological membrane proteins and preparation method of electrophoresis sample

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preparation example Construction

[0053]

[0054] The preparation method of the electrophoretic sample of the present invention utilizes the protein extracted by the above method to prepare the electrophoretic sample, and the obtained electrophoretic sample can obtain a clear electrophoretic spectrum, which is very suitable for electrophoretic analysis, and includes the following steps:

[0055] Take an appropriate amount of protein sample obtained by the biofilm protein extraction method of the present invention, add an appropriate amount of lysate, dissolve with ultrasonic assistance for 20 ± 5 minutes, and vortex for more than 30 minutes until the protein particles cannot be observed with the naked eye, so that the protein is completely dissolved in the lysate centrifuge at 4±1°C for 30±10min at a speed of 13000±1000rpm, take the supernatant to obtain an electrophoresis sample, and the obtained electrophoresis sample can be detected by polyacrylamide gel electrophoresis (SDS-PAGE), or it can be detected by ...

Embodiment 1

[0060] This embodiment provides a method for extracting biofilm proteins, comprising the following steps:

[0061] (1) Take out the ceramsite with biofilm from the filter bed of the earthworm biofilter, wash down the attached biofilm with distilled water, sieve with a sieve of 1 mm aperture to remove impurity particles, and collect the ceramsite under the sieve The material was centrifuged at 4°C for 5 min at a speed of 8000 rpm to collect the biofilm, and it was used as a biofilm sample;

[0062] (2), weigh 4g of biofilm samples into a 50mL centrifuge tube, add 30mL of sodium chloride solution (NaCl) with a concentration of 0.9% (w / v), vortex and shake to wash away the extracellular polysaccharides of microorganisms, and then Centrifuge at 8000rpm for 5min at 4°C to collect the precipitate;

[0063] (3) Wash the precipitate obtained in step (2) with 1×PBS, and after vortexing, centrifuge at 8000 rpm for 5 min at 4°C to collect the precipitate;

[0064] (4) Add 10 mL of lysa...

Embodiment 2

[0073] This embodiment provides a method for preparing an electrophoresis sample, comprising the following steps:

[0074] (1) Pick 80mg of the protein obtained in Example 1 into a 1.5mL centrifuge tube, add 1mL of dissolving solution, place it in an ultrasonic cleaner, and dissolve it with ultrasonic assistance for 20 minutes, then vortex and shake for more than 30 minutes until the protein solid particles cannot be observed with the naked eye Until the protein is completely dissolved in the solution;

[0075] (2) The solution obtained in step (1) was centrifuged at 4° C. for 30 min at a speed of 13,000 rpm, and the supernatant was taken to obtain an electrophoresis sample.

[0076] The above-mentioned electrophoresis sample carries out the step of SDS-PAGE electrophoresis as follows:

[0077] Dilute the electrophoresis sample obtained in step (2) 2-5 times as the sample to be tested for SDS-PAGE electrophoresis, the concentration of the separating gel used is 10%, the conce...

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Abstract

The invention provides an extraction method of biological membrane proteins and a preparation method of an electrophoresis sample. The extraction method comprises the following steps: pretreating a biological membrane sample to obtain a precipitate; mixing the precipitate with a pyrolysis solution and a phenylmethylsulfonyl fluoride solution, and performing ultrasonic pyrolysis under an ice bath condition to obtain a sample pyrolysis solution; and shaking and centrifuging the sample pyrolysis solution, taking a supernatant, and treating the supernatant by adopting a TCA-acetone precipitation method, thereby obtaining a protein sample. The preparation method comprises the following steps: dissolving the protein sample by using a dissolving solution, and sequentially performing ultrasonic treatment, vortex oscillation and centrifugation, and taking a supernatant to obtain the electrophoresis sample. According to the methods provided by the invention, the pyrolysis solution containing various detergents is adopted and is combined with the ice bath ultrasound to break walls of microbial cells, and meanwhile, the TCA-acetone precipitation method is adopted to purify the protein, so that whole proteins in the biological membrane sample can be effectively extracted, and the masking action of humus to the proteins can be reduced to ensure that the analysis result of electrophoresis is clear.

Description

technical field [0001] The invention belongs to the technical field of biological treatment, and relates to a method for extracting biofilm proteins and a method for preparing electrophoresis samples. Background technique [0002] Many studies have proved that the microorganisms identified by traditional isolation and culture methods only account for 1% of the total number of microorganisms in the natural environment. In recent years, the emerging metaproteomics is a new technology that uses proteomic technology to conduct large-scale research and analysis of all proteins produced by a specific microbial community, and it is also a new strategy for exploring the structure of microbial communities. Metaproteome studies can link microbial community composition to their ecological function, leading to a better understanding of functional microbes in a system. [0003] With the continuous deepening and development of sequencing research on the macrogenes of microorganisms and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 邢美燕徐婷王寅卞方杰潘赛孙文杨健吴敏王亚宜
Owner TONGJI UNIV
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