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Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies
- Source :
- Journal of Cellular Biochemistry. 95:990-1001
- Publication Year :
- 2005
- Publisher :
- Wiley, 2005.
-
Abstract
- SIX5 is a member of the human SIX family of transcription factors, many of which are involved in eye development. However, SIX5 transcripts are known to be present at very low levels in cells and no study has yet convincingly demonstrated detection of endogenous SIX5 protein by Western blotting or immunolocalisation. We have produced a new panel of 18 monoclonal antibodies (mAbs) that recognise at least four different epitopes in order to identify authentic human SIX5 protein in cells and tissues. Phage-displayed peptide libraries were used to identify individual amino-acids important for antibody binding within each epitope. Endogenous SIX5 migrated in SDS-PAGE with an apparent M(r) of 100 kDa and was present at similar levels in all foetal tissues and cell lines tested. In HeLa cells, it was located in the nucleoplasm with a granular distribution. An mRNA for a shorter splicing isoform of SIX5, with an altered carboxy-terminus, has been described, but further mAbs specific for this isoform did not detect any endogenous protein. We conclude that the full-length isoform is the major functional protein in vivo while the putative shorter protein is undetectable and may not be expressed at all.
- Subjects :
- Gene isoform
Phage display
medicine.drug_class
Blotting, Western
Molecular Sequence Data
Biology
Monoclonal antibody
Biochemistry
Epitope
Epitopes
Mice
Fetus
Antibody Specificity
Peptide Library
medicine
Animals
Humans
Protein Isoforms
Amino Acid Sequence
RNA, Messenger
Amino Acids
Molecular Biology
Transcription factor
Cell Nucleus
Homeodomain Proteins
Mice, Inbred BALB C
Hybridomas
Sequence Homology, Amino Acid
Alternative splicing
Antibodies, Monoclonal
Cell Biology
Molecular biology
Alternative Splicing
Epitope mapping
Microscopy, Fluorescence
biology.protein
Antibody
Epitope Mapping
HeLa Cells
Subjects
Details
- ISSN :
- 10974644 and 07302312
- Volume :
- 95
- Database :
- OpenAIRE
- Journal :
- Journal of Cellular Biochemistry
- Accession number :
- edsair.doi.dedup.....b33ca31f57e03307172861f14a407b16