Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies.
Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyh1 in mice.
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Xiu-Li
XL
Cao
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Xing
X
Zhang
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Yu-Fei
YF
Zhang
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Yi-Zhe
YZ
Zhang
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Chang-Geng
CG
Song
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Fan
F
Liu
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China.
Yi-Yang
YY
Hu
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China. Electronic address: minhua_zheng@126.com.
Min-Hua
MH
Zheng
State Key Laboratory of Cancer Biology, Department of Medical Genetics and Developmental Biology, The Fourth Military Medical University, Xi'an, 710032, China; Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi'an, 710032, China. Electronic address: huahan@fmmu.edu.cn.
Hua
H
Han
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli; D005260:Female; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D058846:Antibodies, Monoclonal, Murine-Derived; D008565:Membrane Proteins; D011994:Recombinant Proteins; C485951:Ttyh1 protein, mouse
Monoclonal antibody; Neural stem cells; Protein expression; Ttyh1