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.
Figure 2: Linear relationship between protein concentration of lysates from untreated and TNF-α-treated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluence) were treated with TNF-α (10 ng/ml) and lysed after incubation at 37ºC for 5 minutes.
|Product Includes||Volume||Solution Color|
|IkappaBα Mouse mAb Coated Microwells||96 tests|
|Phospho-IkBa (Ser32) Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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.
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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