Product Pathways - PathScan ELISA
PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kit #7173
|7173C||1 Kit (96 assays)||---||In Stock||---|
|7173||carrier free and custom formulation / quantity||email request|
When ordering five or more kits, please contact us for processing time and pricing at email@example.com.
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|Kit Includes||Volume||Solution Color|
|Phospho-NF-κB p65 (Ser536) 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||Colorless|
|STOP Solution #7002||11 ml||Colorless|
|ELISA Wash Buffer (20X)||25 ml||Colorless|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 sheets|
|Cell Lysis Buffer (10X) #9803||15 ml||Yellowish|
Note: 12 8-well modules – Each module is designed to break apart for 8 tests.
Storage: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).
CST's PathScan® Phospho-NF-KappaB p65 (Ser536) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-NF-KappaB p65 protein. A Phospho-NF-KappaB p65 (Ser 536) Mouse mAb has been coated onto the microwells. After incubation with cell lysates, phospho-NF-KappaB p65 protein is captured by the coated antibody. Following extensive washing, NF-KappaB p65 Rabbit mAb is added to detect the captured phospho-NF-KappaB p65 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 optical density for this developed color is proportional to the quantity of phospho-NF-KappaB p65 (Ser536).
Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-NF-KappaB p65 (Ser536) Sandwich ELISA Kit detects endogenous levels of phospho-NF-KappaB p65 at Ser536. As shown in Figure 1, using the Phospho-NF-KappaB p65 (Ser536) ELISA Kit #7173, a significant induction of Phospho-NF-KappaB p65 (Ser536) in HeLa cells treated with TNF-alpha is detected. However, levels of NF-KappaB p65 (either untreatead or treated), detected by the Total NF-KappaB p65 Sandwich ELISA Kit #7174, remain unchanged. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
ELISA - Western correlation
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 #3033 (right panel) or NF-KappaB p65 Antibody #3034 (left panel), is shown in the bottom figure.
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).
- Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
- Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
- Haskill, S. et al. (1991) Cell 65, 1281-9.
- Thompson, J.E. et al. (1995) Cell 80, 573-82.
- Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
- Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
- Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
- Chen, Z.J. et al. (1996) Cell 84, 853-62.
- Senftleben, U. et al. (2001) Science 293, 1495-9.
- Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
- Xiao, G. et al. (2001) Mol Cell 7, 401-9.
- Peiser, M. et al. (2008) J Leukoc Biol , .
- Kubota, S. et al. (2011) Invest Ophthalmol Vis Sci 52, 9142-8. Applications: ELISA.
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- 7174 PathScan® Total NF-κB p65 Sandwich ELISA Kit
- 7834 PathScan® Phospho-NF-κB p65 (Ser536) Sandwich ELISA Antibody Pair
- 7836 PathScan® Total NF-κB p65 Sandwich ELISA Antibody Pair
- 3033 Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb
- 7074 Anti-rabbit IgG, HRP-linked Antibody
- 9803 Cell Lysis Buffer (10X)
- 7004 TMB Substrate
- 7002 STOP Solution
For Research Use Only. Not For Use In Diagnostic Procedures.
PathScan® is a trademark of Cell Signaling Technology, Inc.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.