[Preparation of rabbit antibody against human Id3 and identification of intracellular localization of Id3 in tumor cells].
OBJECTIVE
To express and purify the recombinant human inhibitor of differentiation 3 (Id3) in E.coli and prepare rabbit polyclonal antibody against Id3.


METHODS
The expression vector Id3/pET32a was transformed into E.coli BL21(DE3) and expression of histidine (His)-tagged Id3 fusion protein was induced with IPTG and confirmed by SDS-PAGE and Western blot techniques. Id3 fusion protein was purified through immolbilized Ni(2+); absorption chromatographic column.The purified protein was used to immunize rabbits.The titer and specificity of rabbit antisera were evaluated by immunodouble gel diffusion, ELISA and Western blot techniques. The collected rabbit antisera were purified with polypeptide affinity column. The intracellular distributions of Id3 expression in human human breast cancer cells (MCF7), prostate cancer cells (PC-3M) and lung adenocarcinoma cells (A549) were observed by indirect immunofluorescence assay(IFA).


RESULTS
Id3 fusion protein with 6xhis-tag was successfully expressed in E.coli BL 21. The expressed Id3 with the relative molecular mass size of 34 kD was purified specifically through affinity chromatographic column and confirmed by Western blot. The titers of the rabbit anti-Id3 polyclonal antibodies were 1:8 by immunodouble gel diffusion and 1:8 000 by ELISA. Western blot revealed that the prepared antisera reacted specifically with Id3. IFA showed that in A549 , MCF7 and PC-3M cells, Id3 was mainly distributed in nucleoplasm and the Id3 expression level was significantly higher in MCF7 and PC-3M than in A549 cells.


CONCLUSIONS
The expression and purification of human Id3 protein and preparation of polyclonal antibody against Id3 provide useful tools for the further study of Id3.
Li
Xiao-Jun
XJ
Institute of Clinical Laboratory Medicine, Jinling Hospital, Nanjing 210002, China. lixiaojun62@yahoo.com.cn
Zhong
Ai-Fang
AF
Wang
Kai
K
Jia
Li
L
Chen
Fang-Fang
FF
Yu
Wei
W
D000818:Animals; D000906:Antibodies; D000917:Antibody Formation / Q000276:immunology*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor / Q000378:metabolism / Q000473:pathology*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D051796:Inhibitor of Differentiation Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D009363:Neoplasm Proteins / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits
D000906:Antibodies; D051796:Inhibitor of Differentiation Proteins; D009363:Neoplasm Proteins; C075640:ID3 protein, human