Figure 1: Treatment of HeLa cells with TNF-alpha stimulates phosphorylation of NF-KappaB p65 at Ser536, detected by PathScanTM Phospho-NF-KappaB p65 (Ser536) Sandwich ELISA kit #7173, but does not affect the level of total NF-KappaB p65 detected by PathScanTM Total NF-KappaB p65 Sandwich ELISA kit #7174. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-NF-KappaB p65 (Ser536) (93H1) Rabbit mAb #3034 (right panel) or NF-KappaB p65 Antibody #3033 (left panel), is shown in the bottom figure.
Figure 2: The relationship between protein concentration of lysates from untreated and TNF-alpha-treated HeLa cells and kit assay optical density readings. After starvation, HeLa cells (85% confluence) were treated with TNF-alpha (10 ng/ml) for 7 min at 37oC, and then lysed.
|Product Includes||Volume||Solution Color|
|NF-kB p65 Mouse mAb Coated Microwells||96 tests|
|NF-κB p65 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® Total NF-kappaB p65 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total NF-kappaB p65 protein. A NF-kappaB p65 antibody has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-NF-kappaB p65 proteins are captured by the coated antibody. Following extensive washing, NF-kappaB p65 Antibody is added to detect both the captured phospho- and nonphospho-NF-kappaB p65 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 optical density for this developed color is proportional to the quantity of total NF-kappaB p65 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® Total NF-kappaB p65 Sandwich ELISA Kit detects endogenous levels of NF-kappaB p65 enzyme. As shown in Figure 1, using the Phospho-NF-kappaB p65 (Ser536) Sandwich ELISA Kit #7173, a significant induction of Phospho-NF-kappaB p65 (Ser536) in HeLa cells treated with TNF-alpha is detected. However, the level of NF-kappaB p65 (either untreated or treated), detected by the Total NF-kappaB p65 Sandwich ELISA Kit #7174, 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.
Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).
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