[Preparation and preliminary application of a rabbit anti-human polyclonal antibody against NH2-terminal peptides of RAI16].
OBJECTIVE
To prepare a rabbit anti-RAI16 polyclonal antibody and then to identify and apply the rabbit polyclonal antibody against NH2-terminal peptides of RAI16.


METHODS
The peptide (aa.44 to 55)of human RAI16 NH2-terminus was synthesized by standard Fmoc.The synthesized peptide was purified by reversed phase high-performance liquid chromatography (RP-HPLC) and cross-linked with keyhole limpet hemocyanin (KLH)by sodium metaperiodate. The rabbits were immunized with the conjugated peptide 4 times(400 mug/rabbit).The polyclonal antibody was purified by Protein G from the collected antiserum. Then it was identified and employed for ELISA, immunohistochemistry and Western blot et cetera.


RESULTS
The NH2-terminal peptide of human RAI16 with the purity of 96% was prepared.The titer of the purified polyclonal antibody was 1:125 000. Western blot results showed that the antibody could recognize the protein with molecular weight of 55 000 in the total lysates of human spleen.


CONCLUSIONS
The polyclonal antibody against NH2-terminal peptide generated and identified in the present research can correspond specifically to natural RAI16 protein and therefore it can be employed in ELISA, immunohistochemistry, immunoprecipitation and immunoblotting. The successful preparation of the polyclonal antibody is helpful not only for the study of the tissue distribution and subcellular localization of RAI16, but lso for the research into the function and mechanism of human RAI16.
Luo
Xi-zhong
XZ
Cancer Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China.
Wang
Ge
G
Xu
Wen
W
Li
Qiong
Q
Chen
Chuan
C
Zhang
Zhi-min
ZM
Hu
Qing
Q
Wang
Dong
D
D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011506:Proteins / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits
D000906:Antibodies; D010455:Peptides; D011506:Proteins; C547416:FHIP2B protein, human