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Generation and characterization of polyclonal antibodies specific for human p110 sEGFR
Trace A Christensen1, Jill L Reiter, Andre T Baron
1Tumor Biology Program, Mayo Clinic, Rochester, MN 55905, USA.
Abstract:
The EGFR/ERBB family of receptor tyrosine kinases mediates intracellular signal transduction pathways important in the regulation of cell growth, differentiation, and transformation. We previously have reported the cloning and expression of a 3 kb alternative EGFR transcript which encodes a 110 kDa form of the receptor (p110 sEGFR). This receptor isoform is identical to the extracellular region of the full-length 170 kDa EGFR through amino acid 603; in addition, p110 sEGFR contains 78 unique carboxy-terminal amino acids. Here, we report the generation and characterization of polyclonal antisera specific for the unique carboxy-terminal sequence of p110 sEGFR. Polyclonal antisera were generated by immunizing rabbits with synthetic peptides corresponding to peptides contained within the unique carboxy-terminal sequence of p110 sEGFR. Immunoglobulin fractions from antisera which tested positive for immune reactivity to these peptides by ELISA were affinity-purified by protein G and peptide-based chromatography. This affinity-purified immunoglobulin fraction specifically recognizes p110 sEGFR by ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemical methods. No cross-reactivity with full-length p170 EGFR is observed using any of these detection methods. These new polyclonal antibodies will be useful in determining the expression, localization, and function of p110 sEGFR, and importantly will allow us to distinguish between the expression of this receptor isoform and p170 EGFR.
Insights
Researchers developed specific antibodies for a novel 110 kDa epidermal growth factor receptor (EGFR) isoform, p110 sEGFR. These antibodies distinguish p110 sEGFR from the full-length 170 kDa EGFR, enabling detailed studies of its expression and function.
Area of Science:
- Molecular Biology
- Cell Signaling
- Cancer Research
Background:
- The Epidermal Growth Factor Receptor (EGFR) family is crucial for cell growth and transformation.
- An alternative 3 kb EGFR transcript encodes a 110 kDa isoform, p110 sEGFR, with unique C-terminal amino acids.
Purpose of the Study:
- To generate and characterize polyclonal antibodies specific to the unique C-terminal sequence of p110 sEGFR.
- To enable differentiation between p110 sEGFR and full-length 170 kDa EGFR expression.
Main Methods:
- Polyclonal antisera were raised in rabbits using synthetic peptides from the p110 sEGFR unique sequence.
- Immune immunoglobulin fractions were affinity-purified using protein G and peptide chromatography.
- Antibody specificity was confirmed via ELISA, immunoprecipitation, immunoblot analysis, and immunocytochemistry.
Main Results:
- Generated affinity-purified polyclonal antibodies specifically recognize p110 sEGFR.
- No cross-reactivity was observed with the full-length 170 kDa EGFR.
- The antibodies demonstrated high specificity across multiple detection methods.
Conclusions:
- Newly developed polyclonal antibodies are highly specific for the p110 sEGFR isoform.
- These antibodies are valuable tools for investigating the expression, localization, and function of p110 sEGFR.
- The antibodies will facilitate distinguishing p110 sEGFR from p170 EGFR in biological samples.