[Expression and antibody preparation of FKBP38 and its application].
OBJECTIVE
To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses.


METHODS
The N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF.


RESULTS
The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells.


CONCLUSIONS
We successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.
Liu
An-ling
AL
Institute of Genetic Engineering, Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China. aliu@smu.edu.cn
Zhao
Li
L
Ma
Dong-zhu
DZ
Jia
Chun-hong
CH
Li
Ming
M
Wen
Zhi-hua
ZH
Ye
Yong-bin
YB
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D022021:Tacrolimus Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; C471205:FKBP8 protein, human; D011994:Recombinant Proteins; D022021:Tacrolimus Binding Proteins