Product Pathways - PathScan ELISA
PathScan® Total IκBα Sandwich ELISA Kit #7360
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|Kit Includes||Volume||Solution Color|
|IkappaB-alpha (L27H11) mAb Coated Microwells|
|IkappaB-alpha Detection Ab||11 ml||Green|
|Anti-rabbit IgG, HRP-linked Antibody||11 ml||Red|
|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.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
CST's PathScan® Total IκBα Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total IκBα 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, an IκBα Rabbit Antibody is added to detect the captured IκBα 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 total IκBα protein.Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total IkappaB-alpha Sandwich ELISA Kit detects endogenous levels of total IκBα protein. Using PathScan® Phospho-IκBα (Ser32) 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 this Sandwich ELISA Kit #7360, remains unchanged (Figure 1). 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 phopsphorylation 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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- Chen, Z.J. et al. (1996) Cell 84, 853-62.
- Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63.
- Chen, J. et al. (2008) J Cell Mol Med , .
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- 7355 PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit
- 4812 IκBα (44D4) Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.