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Merck KGaA
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01 June 2012
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Detects and quantifies the level of IκBα protein phosphorylated at Ser32 in human cells. IκBα belongs to IκB protein family and is known to modulate NF-kB (nuclear factor-κB) nuclear translocation. It plays an important role in immune and inflammatory responses, cell division and apoptosis.
| Product information | |||
|---|---|---|---|
| Format | 96-well plate | ||
| Form | 96 Tests | ||
| Detection method | Colorimetric | ||
| Species reactivity | human, not mouse, not rat | ||
| Unit definition | One unit is defined as the amount of IκBα pSer32 derived from 40 pg IκBα phosphorylated by IKKα. | ||
| Sensitivity | < 1.0 Units/ml | ||
| Assay range | 1.6-100 Units/ml | ||
| Assay time | 4 h | ||
| Kit contains | IκBα (pSer32) Standard, Standard Diluent Buffer, IκBα Antibody-Coated 96-Well Plate, Rabbit Anti-IκBα (pSer32) Detector Antibody, Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers and a user protocol. | ||
| Store and ship information | |||
|---|---|---|---|
| Storage | +2°C to +8°C | ||
| Ship |
Blue Ice Only
Multiple Toxicity Values, refer to MSDS |
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| Data | |||
|---|---|---|---|
 Known quantities of IκBα (pSer<sup>32</sup>) protein were measured as outlined in the Detailed Protocol and by immunoblotting using an anti-IκBα (pSer<sup>32</sup>) primary antibody and chemiluminescent detection. The ELISA is approximately 2x more sensitive than immunoblotting.  The specificity of this assay for IκBα phosphorylated at Ser<sup>32</sup> was confirmed by peptide competition. The data show that only the phosphopeptide containing the phosphorylated Ser<sup>32</sup> 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.  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.   Samples of known IκBα (pSer<sup>32</sup>) concentration were assayed in replicates of 16 to determine precision within an assay.  Samples were assayed 48 times in multiple assays to determine precision between assays. |
