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Protein chip and kit for detecting protein acetylation level

A technology of protein chips and kits, which is applied in the direction of biological testing, general structural detail detection of gas analyzers, and measuring devices. The effect of high detection efficiency

Active Publication Date: 2017-08-08
SHANGHAI BIOCHIP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no disclosure of related technologies such as protein chips and kits for detecting the level of post-translational acetylation modification of proteins.

Method used

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  • Protein chip and kit for detecting protein acetylation level
  • Protein chip and kit for detecting protein acetylation level
  • Protein chip and kit for detecting protein acetylation level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation method of the protein chip is as follows:

[0038] The 68 kinds of specific antibodies were diluted to 0.5 μg / μl with PBS-15% glycerol, and the antibodies were spotted onto the chip one by one using the chip spotting platform. The diameter of each spot is 200 μm, and the distance between spots is 500 μm; use biotin-labeled BSA (BSA-biotin) and acetylated BSA (Ac-BSA) as positive controls, and use BSA, mouse IgG, and rabbit normal IgG as negative controls; Put them into a 384-well plate in a certain order, and use a chip spotting instrument to spot the chip. Three replicate spots of ginseng, yin ginseng, and capture antibody are all spotted to prepare a protein chip. The 68 specific antibodies are: AML1, AMPK, APE, AR, α-Tubulin, β-Catenin, Bcl-6, Beclin, BRAF, MYB, c-Myc, CREB, CTBP2, DEK, E2F1, E2F2, E2F3 , EGFR, Her2, Her4, ER, EKLF, FEN1, FOXO1, FOXO3, FOXO4, GATA1, GATA2, GATA3, GATA4, GR, HIF-1α, HMGA1, HMGB1, HSP90, IRF2, IRS1, KRAS, Ku70, MDM2, M...

Embodiment 2

[0041] The detection method of the kit based on the protein chip of embodiment one:

[0042] 1. Sample preparation

[0043] ① Test samples: take 10 6 –5x 10 6 Add 100–200 μl protein lysate to cells or 10–40 mg tissue samples, place at 4°C for 30 minutes and shake from time to time, then centrifuge at 10,000 g for 15 minutes at 4°C, collect the supernatant and quantify it to obtain the test sample ( for detection of acetylation signal);

[0044] ②Background sample: take 100 μg of detection sample, add protein labeling buffer and mix to 50 μl, add 2 μl NHS-biotin and react at room temperature for 2 hours to label, then add 1.6 μl labeling stop buffer and react at room temperature for 30 minutes to stop labeling , and then add 450 μl 1xPBS, put it into an ultrafiltration tube, centrifuge at 10,000 g at 4°C for 30 minutes, and store the ultrafiltrate at -20°C to obtain a blank sample (for detecting protein background signals).

[0045] 2. Protein chip hybridization

[0046] ①...

Embodiment 3

[0053] TSA (Trichostatin A) treated cell model

[0054] 1. Experimental principle

[0055] TSA (Trichostatin A) is a reversible HDAC (histone deacetylase, histone deacetylase) inhibitor, which increases the level of acetylation in cells by inhibiting the process of protein deacetylation. The samples were treated with DMSO and TSA respectively, and the DMSO treatment was used as a control sample. The protein chip and kit of the present invention were used to detect the changes in acetylation levels in cells treated with TSA compared with those treated with DMSO to illustrate the detection effect of the present invention.

[0056] 2. Sample preparation

[0057] Gastric cancer cells MKN45 were cultured in RAPI 1640 medium containing 10% FBS (fetal calf serum, fetal bovine serum) to a density of 70%, and were treated with DMSO and 3 μM TSA for 18 hours to collect cell proteins.

[0058] 3. Test method

[0059] a. Take 10 6 For MKN45 cells treated with DMSO and TSA, add 100 μl ...

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Abstract

The invention discloses a protein chip and a kit which can detect post-translational protein acetylation level, the protein chip includes 68 kinds of specific antibodies, and the kit comprises the protein chip with the point-prepared 68 kinds of specific antibodies, and also includes an antiacetyl lysine antibody, a HRP labeled testing second antibody, biotin labeled tyramine, a signal amplification buffer, 30%H2O2, fluorescence conjugated streptavidin, protein lysate, NHS-biotin, a protein labeling buffer, a labeling termination buffer and a super filteration tube. A detection method is as follows: a protein sample is collected, a part of the protein sample is taken for biotin labeling, the other part of the protein sample is not labeled, the part of the protein sample and the other part of the protein sample are respectively hybridized in two reaction chambers in the protein chip, the biotin-labeled protein sample is used for detection of protein background level, the non-biotin-labeled protein sample is used for detection of protein acetylation level, fluorescence signals of the biotin-labeled protein sample and the non-biotin-labeled protein sample are collected to obtain the ratio of acetylation signal / background signal of the sample, and acetylation level differences can be analyzed by comparison of ratio differences of different samples.

Description

technical field [0001] The invention relates to a protein chip and a kit, in particular to a protein chip capable of detecting the post-translational acetylation modification level of a protein, a kit containing the protein chip and a detection method. Background technique [0002] With the first discovery of histone acetylation by scientists such as Phillips and Allfrey in the 1960s, human understanding of protein acetylation has made great progress in the past 50 years. There are two types of protein acetylation modifications. One is the N-terminal acetylation modification during protein translation, which affects protein synthesis, localization and stability. The other is the acetylation modification on the ε-amino group of lysine residues after protein translation. Under the catalysis of acetyltransferase (HAT), the acetyl group of acetyl-CoA is transferred to the lysine of the target protein. This process is also reversibly regulated by histone deacetylases (HDACs). A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68G01N2440/10
Inventor 戚隽毅张春秀周佳菁肖华胜
Owner SHANGHAI BIOCHIP
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