[Preparation and application of polyclonal antibody against Ras-related C3 botulinum toxin substrate 1].
OBJECTIVE
To prepare rabbit polyclonal antibody (pAb) against Ras-related C3 botulinum toxin substrate 1 (Rac1) and identify the functions of the antibody.


METHODS
The Rac1 fragment was amplified from cDNA of human cervical cancer HeLa cells, and then inserted into the vector pET-32a to construct the recombinant plasmid pET-32a-Rac1. The recombinant protein Rac1 was fused with His-tag and the fusion protein His-Rac1 was expressed in E.coli expression system. Thereafter, His-tagged Rac1 was purified to immunize rabbits as immunogen. The titer and specificity of rabbit polyclonal antibody against Rac1 were identified by ELISA, Western blotting and immunohistochemistry, respectively.


RESULTS
The fusion protein His-Rac1 was successfully expressed in E.coli. The pAb could specifically detect Rac1 in human HeLa cells and MCF-7 cells in Western blotting. Moreover, the pAb could specifically recognized Rac1 protein in human kidney tissues.


CONCLUSIONS
The pAb of human Rac1 protein was successfully prepared, which could specifically recognize Rac1 protein in several natural samples.
School of Graduate, Anhui Medical University, Hefei 230032; Department of Antibody Engineering, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Academy of Military Medicine, Beijing 102206, China.
Lili
L
Zhang
Department of Antibody Engineering, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Academy of Military Medicine, Beijing 102206, China.
Lu
L
Hao
Department of Antibody Engineering, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Academy of Military Medicine, Beijing 102206, China.
Wei
W
Zuo
Department of Antibody Engineering, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Academy of Military Medicine, Beijing 102206, China.
Weijia
W
Ma
School of Graduate, Anhui Medical University, Hefei 230032; Department of Antibody Engineering, Beijing Proteome Research Center, State Key Laboratory of Proteomics, Beijing Institute of Radiation Medicine, Academy of Military Medicine, Beijing 102206, China.
Ying
Y
Liu
D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells; D006801:Humans; D007114:Immunization / Q000379:methods*; D007150:Immunohistochemistry; D007668:Kidney / Q000276:immunology / Q000378:metabolism; D061986:MCF-7 Cells; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000906:Antibodies; C107953:RAC1 protein, human; D011993:Recombinant Fusion Proteins; D020830:rac1 GTP-Binding Protein