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PSer86 monoclonal antibody of Tip60, as well as preparation method and application of pSer86 monoclonal antibody

A monoclonal antibody, serine phosphorylation technology, applied in peptide preparation methods, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as lack and inability to carry out research

Active Publication Date: 2012-06-13
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of Tip60 antibodies that can be used to study endogenous phosphorylation in the domestic and foreign markets, and it is impossible to carry out related research

Method used

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  • PSer86 monoclonal antibody of Tip60, as well as preparation method and application of pSer86 monoclonal antibody
  • PSer86 monoclonal antibody of Tip60, as well as preparation method and application of pSer86 monoclonal antibody
  • PSer86 monoclonal antibody of Tip60, as well as preparation method and application of pSer86 monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Production of monoclonal antibody cell line phosphorylated at Serine 86 of Tip60 and preparation of monoclonal antibody phosphorylated at Serine 86 of Tip60

[0035] 1. Synthetic peptide

[0036] A polypeptide sequence phosphorylated at serine 86 of Tip60 is designed, and the polypeptide sequence is coupled with hemocyanin (keyhole limpet hemocyamin, KLH for short) to form a polypeptide coupling protein as an artificial immunogen. At the same time, an identical polypeptide without phosphorylation was designed as a control.

[0037] 2. Immunization of mice

[0038] Take the polypeptide coupling protein and the same volume of Freund's adjuvant, repeatedly suck and push to mix the two evenly, and then immunize Balb / c healthy female mice after forming a dripping water-in-oil emulsion. After 3-6 times of immunization, the mouse serum was collected, and the antiserum titer was detected by ELISA method. If P / N≥2.1 (P means OD of immune serum sample 450 Value min...

Embodiment 2

[0042] Example 2: Identification and Application of Monoclonal Antibody Phosphorylated at Serine 86 of Tip60

[0043] 1. Identification by ELISA method

[0044] Using a synthetic non-phosphorylated Tip60 polypeptide sequence, a polypeptide sequence phosphorylated at Serine 86 of Tip60, a polypeptide sequence phosphorylated at amino acid 90 of Tip60, and a polypeptide sequence of 86th and 90th positions of Tip60 The amino acid phosphorylated polypeptide sequence was used as a control, and enzyme-linked immunosorbent assay (ELISA) was performed. The results show that the monoclonal antibody phosphorylated at Serine 86 of Tip60 can recognize the polypeptide sequence phosphorylated at Serine 86 of Tip60, and can also recognize the polypeptide sequence phosphorylated at the 86th and 90th amino acids of Tip60, but not Non-phosphorylated Tip60 polypeptide sequence, Tip60 90th amino acid phosphorylated polypeptide sequence (see figure 1 ).

[0045] 2. Detection by Western Blot

[...

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Abstract

The invention provides a monoclonal antibody pSer86 of Tip60, as well as the preparation method and the application of the pSer86 monoclonal antibody, and relates to a novel cell strain and the monoclonal antibody. The cell strain is a monoclonal antibody cell strain pSer86-13MAb obtained by phosphorylating the No. 86 serine of Tip60; the monoclonal antibody obtained by phosphorylating the No. 86serine of Tip60 is an antibody generated by the monoclonal antibody cell strain pSer86-13MAb obtained by phosphorylating the No. 86 serine of Tip60; the antibody excreted by the cell strain can be used for detecting ELISA (enzyme linked immunosorbent assay), immunoblotting, co-immunoprecipitation, immunofluorescent staining and the like; and the antibody can identify not only exogenous overexpression phosphorylated Tip60, but also cell endogenous phosphorylated Tip60, provides convenience for developing and researching phosphorylated Tip60, and provides the first monoclonal antibody with Tip60 of which the No. 86 serine is phosphorylated.

Description

technical field [0001] The present invention relates to a new cell line and monoclonal antibody, in particular to a monoclonal antibody cell line capable of producing Tip60 serine phosphorylation at position 86 and a monoclonal antibody against Tip60 serine phosphorylation at position 86 (pSer86) preparation methods and applications. Background technique [0002] Tip60 (Tat Interactive Protein, 60KDa) is a histone acetyltransferase (HAT) in the most well-studied MYST family, mainly present in a stable nucleoprotein stabilization complex composed of at least 18 subunits . In addition to acetylating histones H2A, H3, and H4, Tip60 can also modify a series of non-histone proteins, such as myeloid histoproliferative oncogene c (c-Myc), kinase ATM, E2F transcription factor, etc. However, under certain conditions, Tip60 also represses transcriptional activity. It has been proved that Tip60 protein is involved in cell signaling, gene expression regulation, chromatin remodeling, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40C07K1/22G01N33/577C12R1/91
Inventor 李晓彤陈艳苏勇李阳叶志云
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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