PathScan Sandwich ELISA Kits

PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit #7080

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Applications Key:
Reactivity Key: H = Human  
Species cross-reactivity is determined by Western blot.

Specificity / Sensitivity

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.

Description

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.

ELISA - Western correlation

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.

Sensitivity

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.

Background

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; Ser177 and Ser181 in the activation loop of IKKβ (serine 176 and 180 in IKKα) are the specific sites whose phosphorylation causes conformational changes resulting in kinase activation (10-13).

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Application References

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Companion Products

• 9809 Phosphate Buffered Saline with Tween 20 (PBST-20X)
• 2078 Phospho-IKKα (Ser176)/IKKβ (Ser177) (C84E11) Rabbit mAb
• 2687 Phospho-IKKα/β (Ser176/180) Antibody
• 2697 Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb
• 9998 BSA
• 2694 Phospho-IKKα/β (Ser176/180) Antibody II

This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.