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Zymogram active electrophoresis detection method

A detection method and protease technology, which are applied to measurement devices, material analysis by electromagnetic means, instruments, etc., can solve problems such as expensive instruments, and achieve the effects of high sensitivity, accurate measurement, and elimination of electroconversion steps.

Inactive Publication Date: 2015-04-22
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims at the deficiencies of the prior art, and provides a new method for detecting protease spectrum more rapidly and accurately. By applying this method, the accumulation of protein bands, molecular weight deviation and the disadvantages of using expensive instruments in the traditional substrate gel method can be reduced. Realize high-throughput rapid detection of multiple protease profiles

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Bacillus pumilus protease substrate soaking method activity zymogram detection

[0034] The Bacillus pumilus strain was purchased from China General Microorganism Culture Collection and Management Center, address: Institute of Microbiology, Chinese Academy of Sciences, strain number CGMCC NO: 1.1625.

[0035] Proceed as follows:

[0036](1) Streak-inoculate the purchased Bacillus pumilus on solid medium, activate and cultivate it at 28°C for 3 days, then inoculate it in 5ml liquid LB medium, and culture it with shaking at 28°C and 200rpm for 1 day, Then inoculate in the fermentation medium of 100ml by the inoculum amount of 5% (v / v), under the condition of 28 ℃, 200rpm shake and cultivate 36h, centrifuge at 10000rpm, make fermented liquid;

[0037] The above-mentioned solid medium components are as follows, all in parts by weight:

[0038] 1 part of peptone, 0.5 parts of yeast powder, 1.5 parts of agar, 100 parts of distilled water, pH 7.5-8.0;

[0039] Th...

Embodiment 2

[0048] Example 2: Determination of extracellular protease spectrum of marine vibrio (Vibrio sp.CSH1301) and comparison with the substrate gel method

[0049] The operation steps are the same as in Example 1, except that the distilled water in the culture medium is replaced with seawater, and the fermentation and cultivation time is 72 hours. Marine Vibrio (Vibrio sp.CSH1301) is a strain isolated from seawater of mangroves in Haikou in our laboratory. Control test preparation 12.5% ​​separating gel: 30% gel stock solution 1.6ml, 1.5mol / LTris-HCl (pH8.8) 1.0ml, distilled water 1.0ml, 1% casein 0.4ml, 10% ammonium persulfate 25 μ l, TEMED 2.5 μl. Immediately after mixing, carefully inject the separating gel into the gap of the prepared glass plate, and gently add 1ml of distilled water to the top layer with a dropper to prevent the oxygen in the air from inhibiting the polymerization of the gel. After the polymerization is completed, pour it out Cover with liquid. Prepare a 5%...

Embodiment 3

[0050] Embodiment 3: the mensuration of trypsin Trypsin protein zymogram and compare with substrate gel method

[0051] The operation steps are the same as those in the examples, except that the commercial enzyme preparation purchased from Shanghai Sangong, Trypsin, is used, and the substrate soaking method of the present invention is compared with the substrate gel method. The preparation of the electrophoretic gel for the control test was the same as that in Example 2. It was incubated at 37°C for 2 hours, then stained with Coomassie Brilliant Blue R-250 staining solution for 3 hours, and finally decolorized with a decolorizing solution until the transparent bands were clear. Trypsin bands such as image 3 shown.

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Abstract

The invention provides a zymogram electrophoresis detection method, which belongs to the technical field of biotechnology. The method comprises the steps of: preparing gelatin, adding samples, carrying out electrophoresis detection, washing off the gelatin, reacting and developing, wherein a protease substrate is not added in the gelatin preparation step; and the gelatin is soaked in a 1% substrate solution before development. Accordingly, the method can obviate the influence of the substrate on the protein migration rate in the gelatin, and can be used in detection of various zymograms without needing expensive electric transfer equipment, so that the method has wide application.

Description

technical field [0001] The invention relates to a novel electrophoresis detection method of protease spectrum, which belongs to the technical field of biotechnology. Background technique [0002] Proteases (proteinases), also known as peptidases, are a class of enzymes that can hydrolyze proteins and polypeptides. They are widely found in animals, plants and microorganisms, and perform many different physiological functions. Proteases can be divided into many types according to different criteria. According to the method of hydrolyzing the peptide bond, it can be subdivided into endopeptidase, exopeptidase, transpeptidase, etc. According to the optimum pH, proteases can be divided into acidic proteases, alkaline proteases and neutral proteases. The MEROPS database records information about proteases and peptide inhibitors of proteases. According to the amino acids involved in catalysis in the catalytic center, proteases in the database can be divided into six categories: a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 何海伦陈淑华刘丹杨兴昊黄嘉丰
Owner CENT SOUTH UNIV
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