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Differential protein detection method for the effect of alpinin and cardamonin on serum

A technology of differential protein and serum protein, applied in the field of biochemical research, to achieve the effect of short sample processing time, clear map and simple operation

Inactive Publication Date: 2018-01-23
HAINAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far, there has been no report on the direct interaction between human serum samples and drugs using 2-DE combined with MS technology. Timely secretion at a special moment is a variety of protein phenotypes, and is an important source for seeking disease markers

Method used

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  • Differential protein detection method for the effect of alpinin and cardamonin on serum
  • Differential protein detection method for the effect of alpinin and cardamonin on serum
  • Differential protein detection method for the effect of alpinin and cardamonin on serum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] (1) Take out human serum samples from the -80°C refrigerator, take 10 cases of human serum samples and mix them evenly, draw 1 mL of human serum samples and place them in a PE tube, add 200 μL alpinia, mix evenly, and make the alpinia The final concentration is 2.138×10 - 3 mol / L, constant temperature 37°C, r=280rpm, 30min.

[0057] (2) Remove high-abundance proteins according to the steps of serum albumin scavenger

[0058] a. Add 100 μL of solution A in ice bath to 20 μL of serum sample, shake gently to mix.

[0059] b. Place at -20°C for 90 minutes.

[0060] c. Centrifuge at 15000g for 20min at 4°C.

[0061] d. Carefully suck out the supernatant (albumin-containing part), add 1 mL of ice-bathed solution B to the precipitate, vortex and mix well, and place on ice for 15 minutes.

[0062] e. Centrifuge at 15000g for 20min at 4°C. The supernatant was carefully discarded and the pellet was the serum protein with most of the albumin removed.

[0063] f. Dry natural...

Embodiment 2

[0085] (1) Take out the human serum samples from the -80°C refrigerator, take 10 cases of human serum samples and mix them evenly, draw 1mL of human serum samples and place them in a PE tube, add 200μL cardamomine, mix well, so that the final concentration of cardamonin 2.138×10 - 3 mol / L, constant temperature 37°C, r=280rpm, 30min.

[0086] (2) Remove high-abundance proteins according to the steps of serum albumin scavenger

[0087] a. Add 100 μL of solution A in ice bath to 20 μL of serum sample, shake gently to mix.

[0088] b. Place at -20°C for 90 minutes.

[0089] c. Centrifuge at 15000g for 20min at 4°C.

[0090] d. Carefully suck out the supernatant (albumin-containing part), add 1 mL of ice-bathed solution B to the precipitate, vortex and mix well, and place on ice for 15 minutes.

[0091] e. Centrifuge at 15000g for 20min at 4°C. The supernatant was carefully discarded and the pellet was the serum protein with most of the albumin removed.

[0092] f. Dry natur...

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Abstract

The invention provides a method for detecting differential protein produced by action between alpinetin or cardamonin and serum. The method comprises the following steps: (1) shaking up serum protein and alpinetin or cardamonin with the concentration of 1.0*10<-3>mol / L-3.0*10<-3>mol / L and cultivating at the constant temperature; (2) removing highly-abundant proteins according to the steps of a serum albumin scavenger; (3) quantifying an obtained protein sample by use of the Bradford method; (4) performing isoelectric focusing according to the protein loading quantity of 1300 [mu]g / 24 cm of a rubber strip; (5) transferring for polyacrylamide gel electrophoresis after balancing of the rubber strip; (6) after finish of electrophoresis, performing Coomassie brilliant blue staining, decolorization, storage and scanning analysis; (7) performing enzymolysis and mass spectrum identification on differential protein spots.

Description

technical field [0001] The invention relates to a method for detecting the interaction differential display protein of alpinin or cardamonin and human serum protein by two-dimensional electrophoresis research technology, which belongs to the field of biochemical research. Background technique [0002] Serum albumin is the most abundant protein in plasma, with a content of about 4 g / 100 mL (0.6 mmol L -1 ), accounting for 60% of the total plasma protein, is one of the few non-glycoproteins in plasma proteins, which can combine with many endogenous and exogenous compounds to store and transport them. Most of the small molecules of drugs play a certain role by combining with macromolecules in the organism (such as blood proteins), which is also the main factor determining whether the drug has good bioavailability, pharmacological activity, metabolic stability and low toxicity. After the drug enters the blood, it must be more or less bound to the blood protein. The combined dru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 何文英吴秀丽陈昱学丁国华
Owner HAINAN NORMAL UNIV
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