Expression, purification of recombinant human mitochondrial transcription termination factor 3 (hMTERF3) and preparation of polyclonal antibody against hMTERF3.
In mammalian cells, a family of mitochondrial transcription termination factors (MTERFs) regulates mitochondrial gene expression. Mitochondrial transcription termination factor 3 (MTERF3) is the most conserved member of the MTERF family and a negative regulator of mammalian mitochondrial DNA transcription. To create a specific polyclonal antibody against human MTERF3 (hMTERF3), we first cloned hMTERF3 into prokaryotic expression vector pGEX-4T-1, and GST-hMTERF3 was efficiently expressed in Escherichia coli after induction by IPTG. The expressed GST-tagged hMTERF3 fusion protein was purified by passive electro-elution process and then used to immunize BALB/c mice, we obtained anti-GST-hMTERF3 polyclonal antibody purified by protein A column and determined the sensitivity and specificity of the antibody against human MTERF3 by enzyme-linked immunosorbent assay and Western blot assay. Furthermore, the full-length hMTERF3 protein expressed in human embryonic kidney 293T cells was detected by anti-GST-hMTERF3 in western blot analysis and immunofluorescence staining. Taken together, our results demonstrate the functionality of the mouse anti-GST-hMTERF3 polyclonal antibody which will provide a useful tool for further characterization of hMTERF3.
Department of Biochemistry and Molecular Biology, School of Basic Medicine, Dali University, 112 Wanhua Road, Dali 671000, China. xwailp@163.com
Wei
W
Xiong
Yonghui
Y
Luo
Cuixiang
C
Zhang
Deyong
D
Tan
Shaoyuan
S
Zuo
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000302:isolation & purification; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D024101:Mitochondrial Proteins / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; C558380:MTERF3 protein, human; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors