Method for in-situ detection of separase cracking product
A technique for in situ detection of cleavage products, applied in the field of epigenetics
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Embodiment 1
[0118] Embodiment 1 Experimental scheme design
[0119] Figure 1A and Figure 1B is the hydrolysis schematic diagram of the complex based on RAD21 and the complex based on REC8, wherein, Figure 1A Schematic representation of somatic cohesin complexes (RAD21-based) and proteolysis from metaphase to anaphase transition, three proteolytic fragments are RAD21*N, RAD21*M and RAD21*C; Figure 1B Schematic diagram of the proteolysis of the germline-specific cohesin complex (REC8-based) and prometaphase of the first meiosis and anaphase of the second meiosis. The three proteolytic fragments are REC8*N, REC8 *M and REC8*C.
[0120] Cleavage of cohesin occurs at the centromere during the metaphase-to-anaphase transition. In order to study the epigenomic map of cohesin fragmentation during the meiotic and mitotic cycles, a reagent capable of recognizing full-length cohesin subunits from proteolytic fragments produced by separase is required, which requires recognition of separase Th...
Embodiment 2
[0122] Example 2 Identification of Anti-RAD21 Neoepitope Monoclonal Antibody
[0123] In order to prepare a specific monoclonal antibody that recognizes the COOH terminus produced by the hydrolysis of separase cleavage cohesin RAD21, mice were immunized with polypeptides DREIMR and IEEPSR that simulate the cleavage site, and the corresponding relationship between the polypeptide sequence and the cleavage site is as follows Figure 3A shown in .
[0124] The spleen cells of the immunized mice were fused with the myeloma cells, and the positive clones obtained by screening were transplanted into the peritoneal cavity of the mice. The hybridoma ascites was selected for western blot experiments, and some shorter and longer peptides were used as controls to detect the fusion peptide between the COOH end of the polypeptide near the RAD21 cleavage site and GST.
[0125] Through the above operations, two clones with specificity for the RAD21*N-terminal and two clones with specificity...
Embodiment 3
[0128] Example 3 Identification of Anti-REC8 Neoepitope Monoclonal Antibody
[0129] Using a method similar to that in Example 2, a specific monoclonal antibody recognizing the COOH terminus produced by the hydrolysis of the cohesin REC8 cleaved by the separase was prepared. Mice were immunized with peptides that mimic the cleavage site, and the correspondence between the peptide sequence and the cleavage site is as follows: Figure 4A shown.
[0130]The spleen cells of the immunized mice were fused with the myeloma cells, and the positive clones obtained by screening were transplanted into the peritoneal cavity of the mice. The hybridoma ascites was selected for western blot experiments, and the fusion peptide of the COOH terminal of the polypeptide near the REC8 cleavage site and the GST fusion peptide was screened, and a series of antibodies that specifically recognized REC8*N and REC8*M were found, such as Figure 4B shown. It can be seen from the figure that antibodies...
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