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A kit for detecting the modification ability of protein alkylation modifier peg and using method thereof

A technology of modification ability and modifier, which is applied in the direction of material analysis by observing the influence of chemical indicators, and analysis by making materials undergo chemical reactions, which can solve problems such as unsatisfactory results, troublesome operation, and long operation time. Achieve the effect of being conducive to quantitative use, avoiding waste, and easy to find

Inactive Publication Date: 2016-03-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chang Yuan et al. (1996) used bovine serum albumin (BSA) as a model protein, and used gel electrophoresis to determine the modification ability of various activated PEGs. However, this method is cumbersome to operate, takes a long time to operate, and the results are not ideal, and it cannot be quantified. Ability to detect activated PEGylated proteins

Method used

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  • A kit for detecting the modification ability of protein alkylation modifier peg and using method thereof
  • A kit for detecting the modification ability of protein alkylation modifier peg and using method thereof
  • A kit for detecting the modification ability of protein alkylation modifier peg and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Detection of modification ability of GSH modified by mPEG-5000

[0063] 1. Preparation of reagents

[0064] (1) Modifier: 50g of mPEG-5000

[0065] (2) The kit of the present invention includes the following reagents:

[0066] Protein and modifier reaction Buffer at pH 9.0;

[0067] Glutathione (GSH) powder; before use, prepare a 1mmol / L glutathione solution with pH 9.0 with the above Buffer;

[0068] 3.2mol / LNaH 2 PO 4 ;

[0069] 1.0 mmol / LDTNB.

[0070] 2. Operation steps

[0071] (1) Modifier activation:

[0072] S1. Use anhydrous benzene (3A molecular sieve to remove water) to recrystallize cyanuric chloride twice;

[0073] S2. Dissolve 5.5 g of S1-treated cyanuric chloride in 400 mL of anhydrous benzene containing 10 g of anhydrous sodium carbonate, add 50 g of mPEG-5000, stir overnight at room temperature and filter;

[0074] Take about 400mL of the filtrate and slowly add 600mL of ether under stirring to obtain a white precipitate, continue s...

Embodiment 2

[0083] Example 2 Detection of modification ability of GSH modified by mPEG-10000

[0084] 1. Preparation of reagents

[0085] (1) Modifier: 100g of mPEG-10000;

[0086] (2) The kit of the present invention includes the following reagents:

[0087] Protein and modifier reaction Buffer at pH 9.0;

[0088] Glutathione (GSH) powder; before use, prepare a 1mmol / L glutathione solution with pH 9.0 with the above Buffer;

[0089] 3.2mol / LNaH 2 PO 4 ;

[0090] 1.0 mmol / LDTNB.

[0091] 2. Operation steps

[0092] (1) Modifier activation: the same as in Example 1.

[0093] (2) Follow the steps shown in Table 2:

[0094] Table 2

[0095]

[0096] As shown in Table 2, the absorbance value at the wavelength of 412 nm was measured within 3 min to 6 min after the end of the color reaction, and the blank tube was used for zero adjustment. The result is that the absorbance value of the blank tube is 0.0027, indicating that the modifier has no light absorption at the wavelength o...

Embodiment 3

[0100] Example 3 Reliability and accuracy verification test of the kit for detecting the modification ability of the modifier

[0101] In order to verify the reliability and accuracy of the kit for detecting the modification ability of the modifier, the present invention simultaneously measured the amount of residual amino groups of GSH by TNBS method, calculated the amino group modification rate, and compared the results of the two methods.

[0102] The results are shown in Table 3:

[0103] Table 3 The results of the comparison of the two methods

[0104]

[0105] Table 3 shows that two methods are used for the determination, that is, the method for determining the modification ability of the modifier in this kit is compared with the results for the determination of the amino acid residues in the protein, and the obtained results are very close, indicating that the method is reliable in detecting the modification ability of the modifier. .

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Abstract

The invention discloses a kit for detecting the PEG modification ability of a protein alkylation modifier and a use method thereof. This kit includes positive control glutathione powder, protein and modifier reaction buffer, NaH2PO4 solution, 5,5-dithio-p-dinitrobenzoic acid (DTNB) color development solution. The method of using the kit is to set up two groups of proteins modified by modifiers and proteins not modified by modifiers, respectively use 5,5-dithio-p-dinitrobenzoic acid (DTNB) as the color reagent, and use a spectrophotometer to The absorbance of the two groups of reaction systems at a wavelength of 412nm was measured, and the modification ability of the protein alkylation modifier PEG was detected according to the difference in the absorbance of the two groups. The entire detection process of the kit can be completed within 60 minutes, which is fast and simple, has good stability and long storage period, and has important popularization and application value.

Description

technical field [0001] The invention relates to a detection kit and a method for using the same. More specifically, it relates to a kit for detecting the ability of a protein alkylation modifier to modify PEG and a method for using the same. Background technique [0002] Chemical modification of protein plays a very important role. It makes the structure of protein more complex, its function more perfect, its regulation more refined, and its effect more specific. In the late 1950s, using chemical modification methods to study the relationship between the structure and function of protein molecules became a hot spot in the fields of biochemistry and molecular biology. The purpose of chemical modification of proteins is in biomedicine and biotechnology. In biomedicine, chemical modification can reduce immunogenic immunoreactivity, inhibit the production of immunoglobulin E, etc.; in the field of biotechnology, enzymes can efficiently catalyze in organic solvents after chemic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78
Inventor 陈芳艳王林川冯定远
Owner SOUTH CHINA AGRI UNIV
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