Protein two-dimensional electrophoresis method and application thereof

Abstract

The invention discloses a two-dimensional protein electrophoresis method, which includes isoelectric focusing electrophoresis in the first direction and SDS polyacrylamide gel vertical electrophoresis in the second direction, respectively improving the hydration conditions and electrophoresis conditions of the focusing electrophoresis, including Use the hydration solution to hydrate the rubber strip to obtain a hydrated rubber strip, and the pH of the hydrated rubber strip is 4-7, wherein each 100mL hydration solution contains the following components: 41-42.5g of urea , 15-16g of thiourea, 2-2.2g of CHAPS, 0.9-1.1g of DTT, 2-2.2g of IPG Buffer, 0.002-0.003g of bromophenol blue, and the solvent of the hydration solution is ddH 2 O; The voltage and time of isoelectric focusing electrophoresis are set as follows: S1: 50v, 10h; S2: 250v, 3h; S3: 500v, 3h; S4: 1000v, 1h; S5: 8000v, 3h; S6: 8000v, 6‑ 6.5h. When the method is applied to detect the distribution of whole protein in the stable cell of Bacillus amyloliquefaciens, the two-dimensional electrophoresis pattern has high resolution and good repeatability.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a protein two-dimensional electrophoresis method and its application. Background technique [0002] The antibacterial substances produced by Bacillus have attracted the attention of many experts and scholars. Bacillus antibacterial substances are usually divided into two categories: one is antibiotics synthesized by ribosomes, mainly lantibiotics, including subtilin, subtilosin, ericin, etc.; the other is lipopeptides synthesized by non-ribosomes Antibiotics (lipopeptides), mainly include fengycin, surfactin, iturin, bacillomycin, mycosubtilin, bacilysin, etc. Antimicrobial lipopeptides have a strong killing effect on a variety of Gram-positive and negative bacteria in food, and can quickly inhibit the growth of microorganisms. After eating, they are easily hydrolyzed and digested by proteases in the body without toxic and side effects. They are active under acidic condi...

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Problems solved by technology

[0005] The purpose of the present invention is to provide a protein two-dimensional electrophoresis method and its application, which solves the existing protein two-dimensional electrophoresis method. When analyzing the distribution of the whole protein in the stable phase bacterial cells, especially for Bacillus amyloliquefaciens Analyzing the distribution of whole protein in bacterial cells in the stable phase, there are problems of low resolution and poor repeatability of the two-dimensional electrophoresis pattern

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