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Phosphorylation antibody chip kit for detecting human receptor tyrosine kinase (RTK)

A tyrosine kinase and antibody chip technology, which is applied in the field of phosphorylated antibody chip kit and human receptor tyrosine kinase phosphorylated antibody chip kit, can solve the problems of time-consuming, laborious, poor effect, and high cost. Achieve the effect of overcoming cumbersome operation, less specimen consumption, and improving sensitivity

Active Publication Date: 2017-07-04
RAYBIOTECH INC GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It can be seen that the detection of phosphorylation of receptor tyrosine kinases is cumbersome and costly; in addition, since scientists usually use new phosphorylation sites for research, the preparation of phosphorylated antibodies for specific protein sites is often more expensive than the preparation of general antibodies. More time-consuming and labor-intensive, the effect is not good

Method used

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  • Phosphorylation antibody chip kit for detecting human receptor tyrosine kinase (RTK)
  • Phosphorylation antibody chip kit for detecting human receptor tyrosine kinase (RTK)
  • Phosphorylation antibody chip kit for detecting human receptor tyrosine kinase (RTK)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Screening of the best antibody chip carrier.

[0034] Conventional antibody chips mostly use glass slides as carriers. Because glass slides are fragile and the detection equipment is expensive, they are not suitable for small-scale applications, and it also brings difficulties to the transportation of reagents in the diagnostic field. However, the traditional nitrocellulose membrane is fragile and has a high background, resulting in large fluctuations in results. We screened membranes and glass slides that are not treated with active methods on the market. After a lot of screening, we found that whether it is a membrane or a glass slide, the temperature and the active reagent components on the surface are the key to the effect of spotting.

[0035] (1) Screening of diaphragm

[0036] In order to immobilize the antibody on the surface of the membrane, we screened different methods to treat the membrane, as shown in the table below. Then use a fully automatic...

Embodiment 2

[0058] Example 2: Preparation of the kit.

[0059] The antibody chip kit of the present invention comprises the following components:

[0060] Solid phase carrier: standard membrane or glass slide coated with antibody.

[0061] Wash solution: 20X concentrated wash solution containing Tween 20. 1X washing solution is pH 7.2, containing 0.1% Tween 20, 0.1mol / L phosphate buffer.

[0062] Diluent: 2 vials of 15ml 5X Concentrated Diluent D for sample dilution, 1 vial of 15ml 5X Concentrated Diluent B for antibody and HRP-streptavidin. 1X dilution B is 15mM, PBS buffer solution of pH7.4, solute and its mass concentration or molar concentration or volume concentration in said dilution B are as follows: 0.5% casein, 2-4% sucrose, 150mM NaCl . The 1X dilution D is 15mM, pH6.5 PBS buffer solution, the solute and its mass concentration or molar concentration or volume concentration in the sample dilution solution are as follows: 2-4% sucrose, 150mM NaCl.

[0063] Detection Antibody:...

Embodiment 3

[0081] Embodiment 3: Detect RTK phosphorylation protein with the kit of embodiment 2

[0082] The bottom film is placed in the matching square box. Since there are a plurality of chip lattices distributed on the bottom film of this embodiment, the square box is provided with 8 square grids in this embodiment. Each chip lattice is divided into mutually independent reaction areas. In this embodiment, 8 square boxes are arranged, and 2 ml of blocking solution is added to each square, and then incubated at room temperature for 30 minutes, and then carried out sequentially. Follow the steps below:

[0083] 1. Add sample

[0084] Aspirate the blocking solution in each square, put 100 microliters to 5 ml of the sample diluted with the blocking solution into the square with a membrane, and then shake it on a shaker at room temperature for 1 to 2 hours, or in React at 4°C for 12-18 hours.

[0085] 2. Membrane washing

[0086] Washing solution I cleaning: Aspirate the sample from th...

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Abstract

The invention relates to a phosphorylation antibody chip kit for detecting human RTK. The phosphorylation antibody chip kit for detecting human RTK comprises: a solid-phase vector, which is a standard film or slide coated with a specific antibody; a cleaning solution, which comprises a 20X concentrated cleaning solution of 0.1% Tween-20 and a dilution solution thereof; a sample diluting solution; a dilution solution used for diluting the antibody and HRP-streptavidin; a biotinylated detection antibody mixture; a 300X concentrated fluorescein-streptavidin solution; a sample treating solution, which is a 2X cell lysis buffer; and the specific antibody, which is targeted to 71 proteins. The RTK phosphorylation antibody chip kit provided by the invention can rapidly, simply and accurately determine the situations of phosphorylation of 71 proteins in a RTK pathway through one experiment; and pathway activation in cells can be rapidly detected by monitoring the phosphorylation changes of proteins in an experimental model system without complicated immunoprecipitation and blotting.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a phosphorylated antibody chip kit, in particular to a phosphorylated antibody chip kit of human receptor tyrosine kinase. Background technique [0002] Protein phosphorylation refers to the phosphorylation of intracellular proteins at specific amino acid sites catalyzed by protein kinases under the stimulation of external signals, and the phosphorylated proteins will sequentially activate downstream signaling molecules at various levels, and finally undergo a cascade reaction. achieve biological effects. A protein can be phosphorylated at one or more sites by multiple protein kinases. The EGFR family of cell membrane receptors includes four different proteins: EGFR / ErbB1 / HER1, ErbB2 / HER2, ErbB3 / HER3 and ErbB4 / HER4. Under normal physiological conditions, ErbB receptors play a vital role in regulating the signal transduction of cell proliferation, differentiation, motility and apoptos...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N33/543
CPCG01N33/544G01N33/573G01N2333/91205G01N33/543G01N33/68
Inventor 黄若磐黄若春
Owner RAYBIOTECH INC GUANGZHOU
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