CN111426681A

CN111426681A - Method for rapidly judging protein elution degree in simple protein purification process

Info

Publication number: CN111426681A
Application number: CN201910220278.2A
Authority: CN
Inventors: 李燕芳; 苏少博
Original assignee: Shanxi Really Biotechnology Co ltd
Current assignee: Shanxi Really Biotechnology Co ltd
Priority date: 2019-03-22
Filing date: 2019-03-22
Publication date: 2020-07-17

Abstract

The invention is applicable to the technical field of protein purification, and provides a method for quickly judging the protein elution degree in a simple protein purification process. According to the method, Coomassie brilliant blue G250 solution and protein are used for displaying blue, and the protein concentration can be quickly estimated by comparing the color of the mixture of the Coomassie brilliant blue G250 solution and buffer solution, so that the protein elution degree can be judged.

Description

Method for rapidly judging protein elution degree in simple protein purification process

Technical Field

The invention belongs to the field of protein separation and purification, and particularly relates to a method for quickly judging protein elution degree in a simple protein purification process.

Background

The protein is widely applied to the industries of scientific research, food, medical treatment and the like. No matter the target protein obtained from natural products or engineering strains contains hybrid protein, the target protein with higher purity can be obtained by further purification operation, and the application requirements of various industries can be met.

Currently, a protein purification system is mostly used to detect a protein elution process when a column chromatography technology is used for protein purification, and an elution peak is collected. It is convenient to use, but the equipment is expensive, and the cost is higher. In the simple protein purification process, a peristaltic pump and an injector can be used for loading and eluting the protein, and the protein elution process can be detected by connecting equipment such as an ultraviolet detector, a chromatographic workstation and the like, but the detection equipment has higher cost. Or directly collecting eluate, detecting protein concentration at 280 nm with spectrophotometer, and determining elution degree, but the operation is complicated and the protein dosage is large. Therefore, the method for quickly judging the protein elution degree in the simple protein purification process is developed, so that the cost is saved, and great convenience is brought to research work.

Disclosure of Invention

The invention aims to provide a method for quickly judging the protein elution degree in a simple protein purification process, and aims to reduce the cost and improve the efficiency.

The invention adopts the following technical scheme:

a method for rapidly judging protein elution degree in a simple protein purification process comprises the following steps:

1) preparing a Coomassie brilliant blue G250 solution, namely adding 250100 mg of Coomassie brilliant blue G, 50m L of 95% ethanol, 100m L of 85% phosphoric acid and distilled water to 1000 m L, filtering by using filter paper, and respectively taking 50 mu l of each solution and packing in 200 mu l of centrifuge tubes.

2) The protein chromatographic column is connected with a peristaltic pump or a syringe, and the equilibrium column, the protein sample loading and the eluent elution are carried out. Mu.l of the eluate was mixed with 50. mu.l of Coomassie brilliant blue G250 solution, and 5. mu.l of the elution buffer was mixed with 50. mu.l of Coomassie brilliant blue G250 solution as a control to observe the color change and determine the degree of protein elution. If the solution is blue, the protein elution peak appears, and the deeper the blue color is, the higher the protein concentration is; if the color of the solution is consistent with that of the control or is close to the color of the solution without change in a longer elution time, the protein is completely eluted, and the next elution buffer solution can be replaced or the purification process is finished.

3) After the purification is finished, SDS-PAGE detects each collected liquid, and the target protein is collected by ultrafiltration centrifugation or dialysis.

The invention has the beneficial effects that:

according to the method for rapidly judging the protein elution degree in the simple protein purification process, the Coomassie brilliant blue G250 is used for reacting with the protein, the protein concentration is estimated through color change, the elution degree is judged, convenience and rapidness are realized, and the cost is also saved.

Drawings

FIG. 1 shows the result of the color development of the mixture of Coomassie brilliant blue G250 solution and protein according to the embodiment of the present invention;

FIG. 2 is a SDS-PAGE image of a protein elution pool of an embodiment of the invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific embodiments, which are merely preferred embodiments of the invention, and all equivalent changes in the features and principles described in the claims of the invention are included in the scope of the invention.

The embodiment of the invention relates to a method for quickly judging the protein elution degree when His tag protein is purified by using a His Trap HP purification column and a simple device. The embodiment of the invention is realized by the following technical scheme:

2) The His Trap HP chromatographic column is connected with a peristaltic pump or a syringe, a PBS buffer solution balance column, a His label protein sample is loaded, and the PBS buffer solution is used for eluting and removing impurity-removed protein. Mixing 5 μ l of eluent with 50 μ l of Coomassie brilliant blue G250 solution, mixing 5 μ l of PBS with 50 μ l of Coomassie brilliant blue G250 solution as a control, when the solution color is consistent with the control or is close to the color of the control without change in a longer elution time, replacing 25 mM of imidazole eluent for elution, and collecting protein eluent when the solution color is darker than the control, and when the solution color is consistent with the control or is close to the color of the control without change in a longer elution time, replacing 50mM of imidazole eluent for elution until the elution of 500mM of imidazole eluent is completed. Washing the column with distilled water, and preserving the column with 20% ethanol.

The color development result of the mixture of Coomassie brilliant blue G250 solution and protein is shown in figure 1. Wherein, each sample in the group A is PBS and the sample at the end of PBS elution; the samples in group B are respectively 25 mM imidazole, 25 mM imidazole elution collecting liquid and samples at the end of 25 mM imidazole elution; the samples in group C are respectively 50mM imidazole, 50mM imidazole elution collecting liquid and samples at the end of 50mM imidazole elution; the samples in group D are respectively 200 mM imidazole, 200 mM imidazole elution collecting liquid and 200 mM imidazole samples at the end of elution; the samples in group E were 500mM imidazole, 500mM imidazole eluate, and 500mM imidazole eluate at the end of the elution.

3) After the purification is finished, SDS-PAGE detects each collected liquid, and the target protein is collected by ultrafiltration centrifugation or dialysis. The SDS-PAGE of the protein eluate is shown in figure 2. Wherein M is protein Marker, lane 1 is unpurified His-tagged protein, lanes 2-10 are 25 mM imidazole-eluting pool, 25 mM imidazole-eluting end sample, 50mM imidazole-eluting pool, 50mM imidazole-eluting end sample, 200 mM imidazole-eluting pool, 200 mM imidazole-eluting end sample, 500mM imidazole-eluting pool, 500mM imidazole-eluting end sample.

As shown in fig. 1, when raw material is homogenized, the milled raw material conveying air chute 4 and the homogenizing silo inlet elevator 1 are respectively communicated with the kiln inlet elevator 2 through the two-way valve 3, and when the rotary kiln and the raw material mill normally operate, all the milled raw material and the kiln return ash enter the homogenizing silo. When the rotary kiln is operated and the raw material mill is stopped, kiln dust collected by the dust collecting system directly enters the kiln. Therefore, the material in the raw material homogenizing silo is ensured to be uniform, and the large fluctuation of the value of the raw material entering the kiln due to the circulating enrichment of the kiln dust in the homogenizing silo is avoided.

The basic chemical components and the strength control value of the cement clinker refer to national standard GB/T21372-2008 silicate cement clinker, wherein the test method of the chemical property and the physical property of the cement clinker is as follows:

chemical properties: the chemical properties are carried out according to GB/T176;

physical properties of the cement clinker are ground to 350m together with dihydrate gypsum meeting GB175 regulations in a unified small mill of Ø 500mmX500mm laboratory²/kg+10m²Per kg,80um screen residue (mass fraction)<=4% of what is done after the type I portland cement is made. SO in the produced cement₃The content (mass fraction) should be within the range of 2.0-2.5%. All tests (out of 28d strength) should be completed within 10d after the cement is made.

Note 1: in order to ensure the similar grain composition of the cement, the grain size of the grinding clinker is less than 5 mm.

Note 2: in order to ensure as close as possible the particle composition of the produced cement, it is recommended to check the ball composition of the small mill frequently.

The clinker test results chemical property data are shown in table 1:

TABLE 1

Wherein L OSS is loss on ignition.

Physical property data are shown in table 2:

TABLE 2

The three-day compressive strength value of the silicate clinker prepared by the embodiment can be improved to be more than 37MPa, and the problems that the capacity of the cement industry is seriously excessive, the market competition is increasingly violent, and the market share is strived for by continuously optimizing and improving the product quality of enterprises are solved.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for rapidly judging the protein elution degree in the simple protein purification process is characterized in that: the method comprises the following steps:

1) preparing a Coomassie brilliant blue G250 solution, namely adding 100 mg of Coomassie brilliant blue G, 50m L of 95% ethanol, 100m L of 85% phosphoric acid and distilled water to 1000 m L, filtering by using filter paper, and respectively packaging 50 mu l of the mixture into 200 mu l of centrifuge tubes;

2) connecting a protein chromatographic column with a peristaltic pump or an injector, balancing the column, sampling a protein sample, eluting an eluent, mixing 5 mu l of elution collection liquid with 50 mu l of Coomassie brilliant blue G250 solution, mixing 5 mu l of elution buffer liquid with 50 mu l of Coomassie brilliant blue G250 solution as a contrast, observing color change, judging the protein elution degree, if the solution is blue, indicating that a protein elution peak appears, and if the blue color is darker, the protein concentration is higher; if the color of the solution is consistent with that of the contrast or is close to that of the solution without change in the longer elution time, the protein is completely eluted, and the next elution buffer solution can be replaced or the purification process is finished;