Preparation and application of rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB).
- Author:
Lu YUAN
1
;
Wenhua XU
1
;
Tingting GE
1
;
Huiping ZHOU
1
;
Ling YANG
1
;
Fan YANG
1
;
Changmin NIU
1
;
Ying ZHENG
2
Author Information
1. Department of Histology and Embryology, Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA ResearchSchool of Medicine, School of Medicine, Yangzhou University, Yangzhou 225001, China.
2. Department of Histology and Embryology, Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA ResearchSchool of Medicine, School of Medicine, Yangzhou University, Yangzhou 225001, China. *Corresponding author, E-mail: yzzkl@163.com.
- Publication Type:Journal Article
- MeSH:
Male;
Rabbits;
Animals;
Mice;
Ubiquitins;
Escherichia coli/genetics*;
Isopropyl Thiogalactoside;
Antibodies;
Enzyme-Linked Immunosorbent Assay
- From:
Chinese Journal of Cellular and Molecular Immunology
2023;39(9):846-851
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
