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Method for quantitatively detecting protein acetylation level

A quantitative detection and acetylation technology, applied in biological testing, material inspection products, fluorescence/phosphorescence, etc., can solve the problems of lack of rapid and non-radioactive quantitative detection of protein acetylation level, difficult antibodies, and difficulties in acetylating proteins, etc.

Inactive Publication Date: 2013-04-17
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] However, in the study of post-translational modification of acetylation, protein acetylation modification is difficult to produce antibodies with strong affinity, and the antigen-antibody interaction immunoprecipitation reaction is used to enrich Acetylation of proteins in cells is very difficult; from a chemical point of view, the acetyl group is relatively stable and has not yet found a suitable one, similar to phosphorylation (Molecular & Cellular Proteomics, 2007, 6(9):1656-1665) and sugar Kylation modification (Journal of Proteome Research, 2009, 8(2):662-672) enrichment material based on chemical bonding
Therefore, there is still a lack of practical methods for rapid, non-radioactive quantitative detection of protein acetylation levels.

Method used

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  • Method for quantitatively detecting protein acetylation level
  • Method for quantitatively detecting protein acetylation level
  • Method for quantitatively detecting protein acetylation level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of protein chip

[0028] The bovine serum albumin antibody was spotted on the three-dimensional gel chip using the chip spotting instrument of Beijing Boao Company. The concentration of protein antibody is 200μg / ml, and the instrument maintains 50% humidity. Each chip has a total of 12 matrices. The number of sample points in each matrix is ​​6 rows and 8 columns, a total of 48 points. Each point is sampled once, and the volume of the antibody is about 1nl. 4°C refrigerator for later use.

Embodiment 2

[0029] Example 2 Detection of Chip Specificity Using Different Kinds of Proteins

[0030] (1) Use bovine serum albumin PBS solution with a mass-volume concentration of 3 μg / ml, acetylated bovine serum albumin PBS solution with a mass-volume concentration of 3 μg / ml, and acetylated horse serum with a mass-volume concentration of 3 μg / ml Myoglobin PBS solution, the horse myoglobin PBS solution that mass-volume concentration is 3 μ g / ml, the goat serum PBS solution that volume percentage concentration is 5%, as the sample of next step;

[0031] (2) The protein chip prepared in Example 1 was blocked with 20 μl of goat serum PBS-T solution with a concentration of 5% by volume for 1.5 h, incubated with 20 μl of the sample for 1.5 h, washed 3 times with TBST solution, and dried; Incubate with 20 μl of biotin-labeled detection antibody with a mass-volume concentration of 2 μg / ml PBS solution for 1.5 h, wash with TBST three times, and dry; Streptomycin PBS solution was incubated for 1...

Embodiment 3

[0037] Example 3 Detect the linear range of acetylated bovine serum albumin in the serum of patients with liver cancer, and draw a standard curve

[0038](1) Dilute with phosphate buffer solution to get 10 times diluted liver cancer patient serum as sample diluent, dilute acetylated bovine serum albumin to obtain standard acetylated bovine serum albumin solutions with different concentrations, the concentrations are 0.25 μg / ml, 0.5μg / ml, 0.75μg / ml, 1μg / ml, 2μg / ml, 4μg / ml, 6μg / ml, 8μg / ml, as the next sample;

[0039] (2) The protein chip prepared in Example 1 was blocked with 20 μl of goat serum PBS-T solution with a concentration of 5% by volume for 1.5 h, then incubated with 20 μl of the sample for 1.5 h, washed 3 times with TBST, and dried; 20 μl mass-volume concentration of 2 μg / ml biotin-labeled detection antibody PBS solution was incubated for 1.5 h, washed 3 times with TBST, and dried; 20 μl mass-volume concentration was 10 μg / ml fluorescence-labeled streptomycin PBS sol...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses a method for quantitatively detecting a protein acetylation level. The method particularly comprises the steps of preparing protein chips, drawing standard curves and detecting actual samples. The method overcomes the defects that antibodies have weak affinities and acetylated protein is difficult to enrich efficiently when acetylation antibodies are subjected to immune precipitation enriching directly; the protein acetylation level can be quantitatively detected quickly and simply; the used sample amount is less; the difference comparison among the different samples is facilitated; the study of an acetylation difference spectrogram provides a technology platform for establishing high-flux protein acetylation difference spectral analysis; and a predictive value of an acetylation state for liver cancer metastasis can be investigated.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quantitatively detecting acetylated proteins. Background technique [0002] Protein acetylation modification is an important post-translational modification, which is involved in the regulation of various cellular biological processes, such as gene transcription (Bioessays, 1998,20(8):615-626), cell apoptosis (Molecular cell, 2004.13( 5): 627-638), cell differentiation (The Journal of Immunology, 2008, 181(7): 4545), DNA repair (Journal of Biological Chemistry, 2010, 285(7): 4398), and acetylation modification and tumor The occurrence and development of the disease are also closely related (Science's STKE,2000,19(6):1176). [0003] However, in the study of post-translational modification of acetylation, there are problems such as the difficulty of producing antibodies with strong affinity for protein acetylation modification, and the difficulty of enriching ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/52G01N21/64
Inventor 姚斐娜樊惠芝杨芃原李颖
Owner FUDAN UNIV
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