Method for detecting protein interacting with p53 transcription activating domain in cell

Abstract

The invention discloses a method for detecting intracellular proteins interacting with the p53 transcription activation domain. After the biotinylated His-tag antibody and streptavidin-coated photosensitive microspheres are mixed and reacted, the light signal value of the reaction well is measured by a light-excited chemiluminescence analyzer. p53 transcriptional activation domain interacting protein. The present invention is based on the photochemiluminescent immunoassay technology, by constructing a eukaryotic expression vector and expressing it in cells to obtain a cell lysate containing p53-His recombinant protein, which can make the luminescent microspheres coated with p53 monoclonal antibody The distance between the photosensitive microsphere and the biotinylated His tag antibody is less than 200nm. The method of the invention can be used to screen and detect proteins interacting with the p53 transcriptional activation domain, and has the advantages of simple operation, high signal-to-noise ratio, and high-throughput screening.

Description

technical field [0001] The invention belongs to the field of biological protein detection, and more specifically, the invention relates to a method for high-throughput detection of proteins interacting with p53 transcription activation domains in cells. Background technique [0002] p53 is an important tumor suppressor in the human body, known as the "guardian of the genome". It checks DNA damage in G1 phase, and regulates cellular DNA repair, growth cycle arrest, apoptosis, etc. through transcriptional activity to maintain normal cell function. The wild-type p53 protein consists of 393 amino acids with a protein molecular weight of 53KD, including the following structural domains: N-terminal transcription activation domain, proline-rich region, DNA binding domain, tetramerization domain and C-terminal structureless the basic domain. As the protein most related to human tumors found so far, about 50% of tumors cannot function normally due to the inhibition of negative regu...

AI Technical Summary

Problems solved by technology

Looking at the existing technologies, each has its own advantages and disadvantages: co-immunoprecipitation is a simple and widely used operation for analyzing protein-protein interactions under physiological conditions, but the steps are cumbersome and cannot screen interacting proteins on a large scale; GST-Pull down is an in vitro verification of protein interaction. The commonly used technology, which is highly specific but not suitable for large-scale screening; yeast two-hybrid technology can analyze the interaction between known proteins and can also be used to screen genes of unknown proteins, although this method is suitable for large-scale screening of interaction However, it is prone to false positives; the tandem affinity purification technology overcomes the false positives of the yeast two-hybrid technology and can study the protein interaction network at the cellular level on a large scale, but the TAP tag introduced by this technology may affect The combination of target protein and affinity column, etc.; surface plasmon resonance technology does not require label modification, and has high detection sensitivity, which can detect the interaction between proteins in real time and efficiently. However, although this technology has the advantages of high precision and fast detection speed, it is not easy High requirements, expensive, low general applicability

In short, although these methods can meet the scientific research requirements, they have their own shortcomings to a certain extent, which is not conducive to cost-effective detection of the interaction between p53 transcription activation domain and protein

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