Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit #7355

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Kit Includes Volume Solution Color
IkappaB-alpha (L27H11) mAb Coated Microwells
P-IkappaB-alpha Detection Ab 11 ml
Anti-rabbit IgG, HRP-linked Antibody 11 ml
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

Reactivity Key:  H=Human  M=Mouse
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Description

CST's PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Phospho-IκBα (Ser32) protein. An IκBα Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-IκBα proteins are captured by the coated antibody. Following extensive washing, Phospho-IκBα (Ser32) Antibody is added to detect the captured phospho-IκBα (Ser32) 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-IκBα (Ser32) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Phospho-IκBα (Ser32) Sandwich ELISA Kit detects endogenous levels of Phospho-IκBα (Ser32) protein. As shown in Figure 1, using this Sandwich ELISA Kit #7355, a significant induction of Phospho-IκBα (Ser32) in HeLa cells treated with TNF-α can be detected. However, the level of total IκBα (phospho- and nonphospho-), detected by the Total IκBα Sandwich ELISA Kit #7360, 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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Treatment of HeLa cells with TNF-α stimulates phosphorylation of IκBα at Ser32, detected by PathScan® Phospho-IκBα (Ser32) Sandwich ELISA kit, #7355, but does not affect the level of total IκBα protein detected by PathScan® Total IκBα Sandwich ELISA kit, #7360. Treatment with MG132, a proteasome inhibitor (37ºC for 180 min before TNF-α induction), causes accumulation of phospho-IκBα in control and TNF-α-treated cells, shown in both Sandwich ELISA and Western blot analysis. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-IκBα (Ser32) Ab #9241 (right panel) or IκBα (L27H11) Mouse mAb #7361 (left panel), is shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: Linear relationship between protein concentration of lysates from untreated and TNF-α-treated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluence) were treated with TNF-α (10 ng/ml) and lysed after incubation at 37ºC for 5 minutes.

Background

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state complexed with the inhibitory IκB proteins (1-3). Activation occurs via phosphorylation of IκBα at Ser32 and Ser36 followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB (3-7). IκBα phosphorylation and resulting Rel-dependent transcription are activated by a highly diverse group of extracellular signals including inflammatory cytokines, growth factors, and chemokines. Kinases that phosphorylate IκB at these activating sites have been identified (8).

  1. Baeuerle, P.A. and Baltimore, D. (1988) Science 242, 540-6.
  2. Beg, A.A. and Baldwin, A.S. (1993) Genes Dev 7, 2064-70.
  3. Finco, T.S. et al. (1994) Proc Natl Acad Sci USA 91, 11884-8.
  4. Brown, K. et al. (1995) Science 267, 1485-8.
  5. Brockman, J.A. et al. (1995) Mol Cell Biol 15, 2809-18.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  8. Karin, M. and Ben-Neriah, Y. (2000) Annu Rev Immunol 18, 621-63.

Application References

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Companion Products

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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