Human Total IkB-alpha DuoSet IC ELISA

Standard Curve

The Human Total I kappaB-alpha DuoSet IC ELISA specifically recognizes I kappaB-alpha as shown by Western blot analysis of the protein bound by the capture antibody supplied in the kit.

Lysate prepared from MCF-7 human breast cancer cells was incubated in wells coated with Total I kappaB-alpha Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with Total I kappaB-alpha Detection Antibody. In addition to IgG heavy and light chains, only a single band at 39 kDa corresponding to I kappaB-alpha was detected in captured material, indicating that the ELISA is specific for I kappaB-alpha.Specificity was further demonstrated using cross-reactivity experiments with related I kappaB family members. Recombinant human (rh) I kappaB-beta and rhI kappaB-epsilon were tested at 50 ng/mL and did not cross-react or interfere in the assay.

Quantification of total I kappaB-alpha human cell lysates.

Lysates prepared from MCF-7 human breast cancer, Daudi human Burkitt's lymphoma, and HeLa human cervical epithelial carcinoma cells were quantified with this DuoSet® IC ELISA. The same lysates were also immunoblotted (inset) with anti-I kappaB-alpha monoclonal antibody (Catalog # MAB4299). The DuoSet® IC ELISA results correlate well with the relative amounts of I kappaB-alpha detected by Western Blot.