PathScan Sandwich ELISA Kits

PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276

Item #	Description	List Price	Web Price	QTY	Buy
7276S	PathScan® Inflammation Multi-Target Sandwich ELISA Kit - 1Kit	$833.00	$749.70

Applications	Reactivity	Sensitivity	MW (kDa)	Source
	H M

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

Protocols

7276: Sandwich ELISA,

Specificity / Sensitivity

CST's PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276 detects endogenous levels of six proteins: NF-κB p65, phospho-NF-κB p65 (Ser536), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-IκB-α (Ser32). Differential activation of these proteins can be observed over time in response to various cytokine treatments, as shown in Figure 1. The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA kits**. **See companion products.

Description

CST's PathScan® Inflammation Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of NF-κB p65, phospho-NF-κB p65 (Ser536), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-Stat3 (Tyr705) and phospho-IκB-α (Ser32). These molecules represent convergence points and key regulatory proteins in signaling pathways controlling the stress and inflammation response. Sixteen tests are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits**. Briefly, a capture antibody* has been coated onto the microwells. After incubation with cell lysates, the coated antibody captures the target protein. Following extensive washing, a detection antibody* is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein. *Antibodies in kit are custom formulations specific to kit. **See companion products.

Sequence

Figure 2. Schematic representation of a 96-well plate depicting the color-code of the reagents used to detect endogenous levels of NF-κB p65 (gray; 1 & 2), Phospho-NF-κB p65 (Ser536) (orange; 3 & 4), Phospho-SAPK/JNK (Thr183/Tyr185) (purple; 5 & 6), Phospho-p38 MAPK (Thr180/Tyr182) (yellow; 7 & 8), Phospho-Stat3 (Tyr705) (dark pink; 9 & 10) and Phospho-IκB-α (Ser32) (blue; 11 & 12).

Sandwich ELISA

Figure 1. Treatment of HeLa cells with TNFα/IL-1β (A) or IL-6 (B) induces differential phosphorylation of NF-κB p65 at Ser536, SAPK/JNK at Thr183/Tyr185, p38 MAPK at Thr180/Tyr182, Stat3 at Tyr705 and IκB-α at Ser32 as detected by the PathScan® Inflammation Multi-Target Sandwich ELISA Kit #7276. While dynamic phosphorylation is observed throughout the time course, the level of total NF-κB p65, SAPK/JNK, p38 MAPK, Stat3 and IκB-α remains unchanged as demonstrated by sandwich ELISA and Western analysis. HeLa cells (80-90% confluent) were serum starved and stimulated with IL-6 (100 ng/mL) for 5, 10, 20, 40 and 80 minutes at 37ºC. Alternatively, exponentially growing cultures of HeLa (80-90% confluent) were treated simultaneously for the indicated times at 37ºC with 20 ng/mL TNF-α and 10 ng/mL IL-1β. Lysates were assayed at a protein concentration of 0.5 mg/mL. The absorbance readings at 450 nm are shown as a 3-dimensional representation in the left figure, while the corresponding Western blots are shown in the right figure. The antibodies used for the Western analyses include NF-κB p65 Antibody #3034, Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033, SAPK/JNK (56G8) Rabbit mAb #9258, Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mAb #9255, p38α MAP Kinase Antibody #9218, Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mAb #9216, Stat3 Antibody #9132, Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138, IκB-α Antibody #9242 and Phospho-IκBα (Ser32) (14D4) Rabbit mAb #2859.

Application References

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Companion Products

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.