Figure 2. The relationship between the protein concentration of lysates from CCD-1070Sk cells, serum starved or hIL-1β-treated, and the absorbance at 450 nm is shown. CCD-1070Sk cells (80-90% confluent) were serum starved or treated with hIL-1β #8900 (5 ng/ml, 16 hr) and then lysed.
Figure 1. Treatment of CCD-1070Sk cells with IL-1β stimulates expression of Cox2 as detected by the PathScan® Total Cox2 Sandwich ELISA Kit. CCD-1070Sk cells (80-90% confluent) were serum starved or treated with hIL-1β #8900 (5 ng/ml, 16 hr). The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using Cox2 Antibody #4842 is shown in the bottom figure.
|Product Includes||Volume||Solution Color|
|Cox2 Mouse mAb Coated Microwells||96 tests|
|Cox2 (D5H5) Rabbit Detection mAb||1 ea||Green|
|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 Sample Diluent||25 ml||Blue|
|ELISA Wash Buffer (20X) 9801||25 ml|
|Cell Lysis Buffer (10X) 9803||15 ml|
The PathScan® Total Cox2 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Cox2. A Cox2 Mouse Antibody has been coated onto the microwells. After incubation with cell lysates, Cox2 protein is captured by the coated antibody. Following extensive washing, a Cox2 Rabbit Detection Antibody is added to detect the captured Cox2 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 the absorbance for the developed color is proportional to the quantity of Cox2.
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
PathScan® Total Cox2 Sandwich ELISA Kit detects endogenous levels of total Cox2 protein. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Cyclooxygenase1 (Cox1) and cyclooxygenase2 (Cox2), family members with 60% homology in humans, catalyze prostaglandin production from arachidonic acid (1,2). While Cox1 expression is constitutive in most tissues, Cox2 expression is induced by lipopolysaccharide (LPS) and peptidoglycan (PGN) (3). PGN activates Ras, leading to phosphorylation of Raf at Ser338 and Erk1/2 at Tyr204. The activation of MAP kinase signaling results in subsequent activation of IKKα/β, phosphorylation of IκBα at Ser32/36, and NF-κB activation. Finally, activation of the transcription factor NF-κB is responsible for the induction of Cox2 expression (4). Investigators have shown that LPS and PGN induce the clinical manifestations of arthritis and bacterial infections, such as inflammation, fever, and septic shock (5). Research studies have indicated that Cox1 and Cox2 may also play a role in the neuropathology of Alzheimer's disease by potentiating γ-secretase activity and β-amyloid generation (6).
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