CBA065 PhosphoDetect™ IκBα (pSer³²) ELISA Kit

Table 1: Dilution of IκBα (pSer³²) Standard

Table 2: Guidelines for diluting Anti-Rabbit IgG horseradish Peroxidase (HRP)

Table 3: Example Data

Example data were obtained for the various diluted standards over the range of 0 to 100 Units/ml IκBα (pSer³²).

Figure 1: Sensitivity

Known quantities of IκBα (pSer³²) protein were measured as outlined in the Detailed Protocol and by immunoblotting using an anti-IκBα (pSer³²) primary antibody and chemiluminescent detection. The ELISA is approximately 2x more sensitive than immunoblotting.

Table 4: Intra-Assay Precision

Samples of known IκBα (pSer³²) concentration were assayed in replicates of 16 to determine precision within an assay.

Table 5: Inter-Assay Precision

Samples were assayed 48 times in multiple assays to determine precision between assays.

Figure 2: IκBα (pSer³²) ELISA Parallelism

Natural phospho IκBα from human Jurkat cell lysates was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the IκBα (pSer³²) standard curve. Parallelism was demonstrated by the figure above and indicated that the standard accurately reflects IκBα (pSer³²) content in samples.

Table 6: Linerearity of Dilution

Jurkat cells were grown in tissue culture medium containing 10% fetal calf serum treated with TNF-α for 5 min and lysed with Cell Lysis Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for IκBα (pSer³²) content. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99 in both cases.

Figure 3: Assay Specificity

The data indicate that IκBα (pSer³²) recognizes E. coli-derived recombinant IκBα phosphorylated in vitro by recombinant IKKα subunit. An ELISA measuring total IκBα recognizes both phosphorylated and non-phosphorylated IκBα.

Figure 4: Peptide Competition Assay

The specificity of this assay for IκBα phosphorylated at Ser³² was confirmed by peptide competition. The data show that only the phosphopeptide containing the phosphorylated Ser³² could block the ELISA signal in Jurkat cells treated with TNF-α. The same sequence containing non-phosphorylated serine at position 32 did not block the signal. A peptide conatining a phosphoserine at position 36 also did not block the signal.

Figure 5: Detection of IκBα in Jurkat Cells

Cell lysates made from Jurkat cells were harvested at various times following treatment with TNF-α and compared to non-treated controls, as determined using the Detailed Protocol provided. The data presented here also demonstrate that there is an initial increase in phosphorylation followed by a reduction in the level of Total IκBα which is attributable to the TNF-α-stimulated degradation of IκBα. The results correlate well with immunoblot analysis.