Figure 1. Treatment of differentiated THP-1 cells with LPS stimulates phosphorylation of IKKβ at Ser177/181 as detected by the PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit #7080, but does not affect the level of total IKKβ protein. Differentiated THP-1 cells were treated with 1 μg/ml LPS for 10 minutes. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blots using IKKβ (2C8) Rabbit mAb #2370 (left panel) or Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb #2697 (right panel) are shown in the bottom figure.
Figure 2. The relationship between the protein concentration of lysates from untreated and LPS-treated THP-1 cells and the absorbance at 450 nm using the PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit is shown.
|Product Includes||Volume||Solution Color|
|Phospho-IKK(Ser176/180) Rabbit mAb Coated Microwells||96 tests|
|IKK beta Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse 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|
|PathScan® Sandwich ELISA Lysis Buffer (1X) 7018||30 ml|
The PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of IKKβ when phosphorylated at Ser177/181. A phospho-IKKβ (Ser177/181) rabbit mAb has been coated onto the microwells. After incubation with cell lysates, phospho-IKKβ (Ser177/181) protein is captured by the coated antibody. Following extensive washing, an IKKβ mouse detection mAb is added to detect the captured IKKβ protein. Anti-mouse IgG, HRP-linked antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for the developed color is proportional to the quantity of IKKβ phosphorylated at Ser177/181.
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
revised January 2016
Protocol Id: 205
The PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit detects endogenous levels of IKKβ protein when phosphorylated at Ser177/181, as shown in Figure 1. The kit sensitivity is shown in Figure 2. 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). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).
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