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Method and kit for detecting Ras activity of small molecular G protein by utilizing GST pull-down and Western-Blot

A G protein and small molecule technology, applied in the detection of small molecule G protein Ras activity, methods and kits, can solve the problems of tumor loss, etc., and achieve the effect of inhibiting degradation, simple operation and high purity

Inactive Publication Date: 2016-04-06
TAISHAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When the ras gene mutates into an oncogene, the expression product Ras protein loses the control of the transition between GTP and GDP, and becomes constitutively active Ras-GTP, and the continuous activation of Ras-GTP often leads to the occurrence of tumors

Method used

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  • Method and kit for detecting Ras activity of small molecular G protein by utilizing GST pull-down and Western-Blot
  • Method and kit for detecting Ras activity of small molecular G protein by utilizing GST pull-down and Western-Blot
  • Method and kit for detecting Ras activity of small molecular G protein by utilizing GST pull-down and Western-Blot

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Using this kit to detect the activity levels of N-Ras and K-Ras in PC-12 cells

[0065] The specific implementation steps are as follows:

[0066] 1. Preparation of cell supernatant containing Ras-GTP

[0067] It has been reported that nerve growth factor (NGF, 50ng / mL) stimulated PC-12 cells can cause Ras (H-Ras, K-Ras and N-Ras) activation. In NGF stimulation experiments, cells were seeded at 35 cm 2 In the culture dish, the cells were serum-starved for 16 hours when the cells grew to 80% density. The cells in the treatment group were stimulated with NGF at a final concentration of 50 ng / mL for 5 min, and served as a negative control. After the stimulation, wash the cells once with PBS, use the IPbuffer provided by this kit to lyse the cells on ice, centrifuge at 12,000rpm for 10min, take the supernatant for later use, and keep 20% of it as a control.

[0068] 2. According to the method 1-5 described in the kit of the present invention, process the glut...

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Abstract

The present invention belongs to the field of molecular biology, and discloses a method and kit for detecting Ras activity of a small molecular G protein by utilizing GST pull-down and Western-Blot. The kit comprises: a conventional GST pull-down experimental components, a GST-Raf1-RBD fusion protein in specific binding with Ras-GTP, and an antibody for specific recognition of three Ras subtypes (H-ras, K-ras and N-ras). In the detection of Ras activity, the method and kit for detecting Ras activity of a small molecular G protein have the advantages of rapidness, accuracy and simpleness, and is conducive to clinical and research application.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method and a kit for detecting the activity of small molecule G protein Ras. Background technique [0002] The ras gene is a proto-oncogene, named after it was first discovered in rat sarcoma virus, and its encoded protein is the small molecule G protein Ras. In cell signal transduction, under the action of guanylate replacement factor, inactive Ras-GDP can be transformed into active Ras-GTP, which participates in cell proliferation and differentiation by starting the MAP kinase pathway in signal transduction. This gene is frequently mutated in human cancer cells. [0003] There are three characteristic genes related to human tumors in the ras gene family, namely H-ras, K-ras and N-ras, which are located on human chromosomes 11, 12 and 1 respectively, and the encoded Ras protein is composed of 188 or 189 amino acids with a molecular weight of 21kDa. Ras protein has high spec...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/70C12P21/02C07K19/00
CPCC07K14/4722C07K2319/23C12N15/62C12N2800/101G01N33/68
Inventor 张宗勇孙保亮杨明峰李大伟
Owner TAISHAN MEDICAL UNIV
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