Extraction method of biofilm protein and preparation method of electrophoresis sample
An extraction method and biofilm technology, applied in the preparation of test samples, etc., can solve the problems of stagnation in macroproteomics environmental research, difficulty in sample purification, and difficulty in sample analysis, so as to reduce the interference of electrophoresis and clear protein spots , The effect of simple operation process
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[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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