CBA038 PhosphoDetect™ Insulin Receptor (pTyr^1162/1163) ELISA Kit

Table 1: Dilution of IR (pTyr^1162/1163) Standard

Discard all remaining reconstituted and diluted standards after completing assay. Return the Standard Diluent Buffer to the refrigerator.

Table 2: Example Anti-rabbit IgG-HRP Working Solution

Table 3: Typical Data Obtained with Standard

The above data was obtained for the various standards over the range of 0 to 100 Units/ml IR (pTyr^1162/1163).

Figure 1: Sensitivity

The sensitivity of this ELISA was compared to immunoblotting using cell lysate with known quantities IR (pTyr^1162/1163). The data presented in Figure 1 show that the sensitivity of the ELISA is about 2x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using rabbit anti- IR (pTyr^1162/1163), and chemiluminescent detection.

Table 4: Intra-Assay Precision

Samples of known STAT1 (pTyr⁷⁰¹) 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: Parallelism

Natural IR (pTyr^1162/1163) from lysate of insulin-stimulated CHO-T cells was serially diluted in Standard Diluent Buffer. The optical density of each dilution was plotted against the human IR (pTyr^1162/1163) standard curve. Parallelism was demonstrated by the figure below and indicated that the Standard accurately reflects IR (pTyr^1162/1163) content in samples.

Table 6: Linearity of Dilution

Lysate Buffer was spiked with IR (pTyr^1162/1163) and serially diluted in Standard Diluent Buffer over the range of the assay. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.

Figure 3: Specificity

This IR (pTyr^1162/1163) ELISA kit is specific for measurement of IR that is dually phosphorylated at Tyr^1162/1163. IR of natural or recombinant (β-subunit) origin is reactive in this assay. This kit detects phosphorylated IR in insulin-stimulated CHO-T cells and does not detect non-phosphorylated IR in unstimulated cells, as shown in figure 3.

Figure 4: Specificity

The specificity of this assay for IR phosphorylated at Tyr^1162/1163 was confirmed by peptide competition. Phosphorylated IR was quantitated in the assay as usual except that the detection antibody was preincubated with IR-derived peptides at a concentration of 0.1-1 µg/ml. The data presented in Figure 3 show that only the peptide corresponding to the region surrounding Tyr^1162/1163, containing the phospho-tyrosines, could block the ELISA signal. The peptides containing phosphorylated tyrosine at positions 972 and 1158 did not block the signal.

Figure 5: Specificity

Figure 5 shows that IR (pTyr^1162/1163) phosphorylation in CHO-T cells is dependent on levels of insulin stimulation. Cells (~90% confluent) were treated with insulin at varying concentrations (0-100 nM) for 10 min, lysed and quantitated in parallel for IR content (both β-subunit and pTyr^1162/1163). The amount of IR (β-subunit) remains constant, while the level of phosphorylation at Tyr^1162/1163 decreases with diminishing insulin dosage. Phosphorylation at IR pTyr¹¹⁵⁸, shows a similar pattern.