The polyclonal antibodies against BCAR1, raised in chickens (328#) and rabbits (329#), were used diluted 300- to 10 000-fold.
BCAR1 concentrations were measured by ELISA with the same experimental setup as described previously by Grebenschikov et al.
Purified His-BCAR1/F1R5 was used as a calibrator in the BCAR1 ELISA to generate the dose-response curve.
Assay specificity was checked by analyzing different dilutions of four cytosol preparations based on ZR-75-1 breast cancer cell-derived stable transfectants (ZR-75-1, F-BCAR1, BCAR1, and F-Hybrid).
To develop antibodies specific for BCAR1, we selected a central part of the BCAR1 cDNA that encoded residues 272-541 of the protein (Fig.
The sodium dodecyl sulfate--polyacrylamide gel was loaded with total extracts from ZR-75-1 breast cancer cells together with ZR-75-1 cell-derived stable transfectants that overproduced BCAR1 or HEF1, and with in-vitro-produced BCAR1 and HEF1 (ITT BCAR1 and ITT HEM).
In addition to FLAG-tagged BCAR1 (ITT F-BCAR1) and HEF1 (ITT HEM), we used FLAG-tagged chimerical protein of BCAR1/HEF1 (ITT F-Hybrid) and BCAR2/Tree-132 protein as a negative control (ITT Control).
The preparation His-BCAR1/F1R5 was used as a calibrator to generate the dose-response curve of the BCAR1 ELISA.
To monitor long-term performance of the assay, we measured BCAR1 protein concentrations in three reference preparations included in all assays performed during the study.
The lowest concentration of BCAR1 protein (-22 mg/kg protein) was in the cytosol of the parental cell line ZR-75-1 and in the cytosol preparation of the transfectant that overproduced FLAG-tagged hybrid molecule (F-Hybrid).
4B) showed that only in the preparation with FLAG-tagged BCAR1 production (ITT F-BCAR1) was a high concentration of the analyte found, whereas in both the ITT HEM and ITT F-Hybrid preparations the concentrations of BCAR1 were very close to that of the negative control protein preparation (ITT Control).