Product Pathways - PathScan ELISA
PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit #7355
|7355S||1 Kit (96 assays)||---||In Stock||---|
|7355||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
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|Kit Includes||Volume||Solution Color|
|IkappaB-alpha (L27H11) mAb Coated Microwells|
|P-IkappaB-alpha Detection Ab||11 ml|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml|
|TMB Substrate #7004||11 ml||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|Sealing Tape||2 sheets|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
CST's PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-IκBα (Ser32) protein. An IκBα Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-IκBα proteins are captured by the coated antibody. Following extensive washing, Phospho-IκBα (Ser32) Antibody is added to detect the captured phospho-IκBα (Ser32) protein. Anti-rabbit IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of phospho-IκBα (Ser32) protein.
Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit detects endogenous levels of Phospho-IκBα (Ser32) protein. As shown in Figure 1, using this Sandwich ELISA Kit #7355, a significant induction of Phospho-IκBα (Ser32) in HeLa cells treated with TNF-α can be detected. However, the level of total IκBα (phospho- and nonphospho-), detected by the Total IκBα Sandwich ELISA Kit #7360, remains unchanged. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
ELISA - Western correlation
Figure 1: Treatment of HeLa cells with TNF-α stimulates phosphorylation of IκBα at Ser32, detected by PathScan® Phospho-IκBα (Ser32) Sandwich ELISA kit, #7355, but does not affect the level of total IκBα protein detected by PathScan® Total IκBα Sandwich ELISA kit, #7360. Treatment with MG132, a proteasome inhibitor (37ºC for 180 min before TNF-α induction), causes accumulation of phospho-IκBα in control and TNF-α-treated cells, shown in both Sandwich ELISA and Western blot analysis. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-IκBα (Ser32) Ab #9241 (right panel) or IκBα (L27H11) Mouse mAb #7361 (left panel), is shown in the bottom figure.
The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins (1-3). Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified (8).
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- Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
- Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
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- Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63.
- Hirsch, H.A. et al. (2010) Cancer Cell 17, 348-61. Applications: ELISA.
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For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.