Product Pathways - PathScan ELISA
PathScan® Total IκB-α Sandwich ELISA Kit #7360
| Kit Includes | Volume | Solution Color |
|---|---|---|
| IkappaB-alpha (L27H11) Mouse mAb Coated Microwells | ||
| IkappaB-alpha Detection Ab | 11 milliliters | Green |
| Anti-rabbit IgG HRP-Linked Ab | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | 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 (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H M
Reactivity Key: H=Human M=Mouse
Description
CST's PathScan® Total IkappaB-alpha Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total IkappaB-alpha protein. An IkappaB-alpha Mouse mAb 7361* has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-IkappaB-alpha proteins are captured by the coated antibody. Following extensive washing, an IkappaB-alpha Antibody 7362* is added to detect the captured IkappaB-alpha protein. HRP-linked anti-rabbit antibody #7074* 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 IkappaB-alpha 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 IkappaB-alpha protein. Using PathScan® Phospho-IkappaB-alpha (Ser32) Sandwich ELISA Kit #7355, a significant induction of phospho-IkappaB-alpha (Ser32) in HeLa cells treated with TNF-alpha can be detected. However, the level of total IkappaB-alpha (phospho- and nonphospho-), detected by this Sandwich ELISA Kit #7360, remains unchanged (Figure 1).
Western Blotting
Figure 1: Treatment of HeLa cells with TNF-alpha stimulates phopsphorylation of IkappaB-alpha at Ser32, detected by PathScan® Phospho-IkappaB-alpha (Ser32) Sandwich ELISA kit, #7355, but does not affect the level of total IkappaB-alpha protein detected by PathScan® Total IkappaB-alpha Sandwich ELISA kit, #7360. Treatment with MG132, a proteasome inhibitor, (37oC for 180 min before TNF-alpha induction) causes accumulation of phospho-IkappaB-alpha in control and TNF-alpha-treated cells, shown in both Sandwich ELISA and Western blot analysis. OD450 readings are shown in the top figure, while the corresponding Western blot using Phospho-IkappaB-alpha (Ser32) Ab #9241 (right panel) or IkappaB-alpha (L27H11) Mouse mAb #7361 (left panel), is shown in the bottom figure.
Sandwich ELISA
Figure 2: Linear relationship between protein concentration of lysates from untreated and TNFalpha-treated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluence) were treated with TNFalpha (10 ng/ml) and lysed after incubation at 37oC for 5 minutes.
Background
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 IκB-α at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκB-α 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). Because phosphorylation of IκB-α at Ser32/36 is essential for release of active NF-κB, phosphorylation at this site is an excellent marker of NF-κB activation (1-3).
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Application References
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