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 | 2 to 8°C |  | Blue ice |  | Multiple Toxicity Values, refer to MSDS | | Note: Store and Ship conditions may differ. | | See Key |
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| | PhosphoDetect™ IkBa (pSer32) ELISA Kit |
|  | Cat. No. CBA065 |
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| Format: 96-well plate | | Form: 96 Tests | | Detection Method: Colorimetric | | Species Reactivity: human, not mouse, not rat | | Sensitivity: < 1.0 Units/ml | | Assay Range: 1.6-100 Units/ml | | Unit Definition: One unit is defined as the amount of IkBa pSer32 derived from 40 pg IkBa phosphorylated by IKKa. | | Assay Time: 4 h | | Kit Contents: IkBa (pSer32) Standard, Standard Diluent Buffer, IkBa Antibody-Coated 96-Well Plate, Rabbit Anti-IkBa (pSer32) Detector Antibody, Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers and a user protocol. | | Comments: Detects and quantifies the level of IkBa protein phosphorylated at Ser32 in human cells. IkBa belongs to IkB protein family and is known to modulate NF-kB (nuclear factor-kB) nuclear translocation. It plays an important role in immune and inflammatory responses, cell division and apoptosis. | | Ref.: Ma, M.H., et al. 2003. Clin. Cancer Res. 9, 1136. Ghosh, S. and Karin, M. 2002. Cell 109, S81. Adams, J. 2001. Semin. Oncol. 28, 613. Berenson, J.R., et al. 2001. Semin. Oncol. 28, 626. Joyce, D., et al. 2001 Cytokine Growth Factor Rev. 12, 73. Tanaka K., et al. 2001. Biochimie 83, 351. Israel, A. 2000. Trends Cell Biol. 10, 129. Malek, S., et al. 1998. J. Biol. Chem. 273, 25427. |
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Detection of IkBa in Jurkat Cells. Cell lysates made from Jurkat cells were harvested at various times following treatment with TNF-a 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 IkBa which is attributable to the TNF-a-stimulated degradation of IkBa. The results correlate well with immunoblot analysis.
Guidelines for diluting Anti-Rabbit IgG horseradish Peroxidase (HRP).
Inter-Assay Precision. Samples were assayed 48 times in multiple assays to determine precision between assays.
Intra-Assay Precision. Samples of known IkBa (pSer32) concentration were assayed in replicates of 16 to determine precision within an assay.
Peptide Competition Assay. The specificity of this assay for IkBa 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-a. 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.
Sensitivity. Known quantities of IkBa (pSer32) protein were measured as outlined in the Detailed Protocol and by immunoblotting using an anti-IkBa (pSer32) primary antibody and chemiluminescent detection. The ELISA is approximately 2x more sensitive than immunoblotting.
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