Immunodetection of the presence or absence of full-length APC gene product in human colonic tissues.
In order to detect the presence or absence of wild-type adenomatous polyposis coli (APC) gene protein (APC) in human colonic tissues, we immunoaffinity purified two polyclonal rabbit antibodies (APC-1 and APC-2) directed against defined epitopes in the middle and carboxyl regions of APC. Such antibodies proved useful in western blot analysis of matched colonic mucosa and tumor sample pairs. A 300 kDa band corresponding to APC was detected in samples from normal colonic mucosa using both antibodies. No tumor samples (n = 14) showed a detectable 300 kDa band. SW480 colon carcinoma cells, known to express truncated APC lacking the carboxyl half of the protein, were also negative. These results indicate that our antibodies bind to full-length but not truncated APC. Thus, western blot analysis employing APC-1 and APC-2 antibodies may be used to evaluate the absence or presence of wild-type APC. The value of this methodology in detecting APC mutations, which mainly involve protein truncation or allelic loss, is based on its ability to demonstrate negative or reduced level of immunoreactivity toward full-length APC in tissues that contain such mutations.
Chop
A M
AM
Creighton Cancer Center, Creighton University School of Medicine, Omaha, NE 68178, USA.
Abraham
C L
CL
Adrian
T E
TE
Murphy
R
R
Boman
B M
BM
D000230:Adenocarcinoma / Q000235:genetics / Q000473:pathology / Q000601:surgery; D000236:Adenoma / Q000235:genetics / Q000473:pathology / Q000601:surgery; D011125:Adenomatous Polyposis Coli / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D025601:Adenomatous Polyposis Coli Protein; D000906:Antibodies; D015153:Blotting, Western; D002460:Cell Line; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology / Q000601:surgery; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D016147:Genes, Tumor Suppressor; D006801:Humans; D007413:Intestinal Mucosa / Q000473:pathology; D011863:Radioimmunoassay; D014407:Tumor Cells, Cultured
D025601:Adenomatous Polyposis Coli Protein; D000906:Antibodies; D003598:Cytoskeletal Proteins