Revision 6

#7360

Store at +4C

PathScan® Total IkappaB alpha Sandwich ELISA Kit

1 Kit

(96 assays)

Species Cross Reactivity:
H M

UniProt ID:
#P25963

Entrez-Gene Id:
#4792

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes Product # Quantity Color Storage Temp
IκBα Mouse mAb Coated Microwells3518396 tests+4C
IkBa Rabbit Detection mAb141521 eaGreen (Lyophilized)+4C
Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)132721 eaRed (Lyophilized)+4C
Detection Antibody Diluent1333911 mlGreen+4C
HRP Diluent1351511 mlRed+4C
TMB Substrate700411 ml+4C
STOP Solution700211 ml+4C
Sealing Tape545032 ea+4C
ELISA Wash Buffer (20X)980125 ml+4C
ELISA Sample Diluent1108325 mlBlue+4C
Cell Lysis Buffer (10X)980315 ml-20C

Kit contents scale proportionally with size, except sealing tape.
Example: The V1 kit contains 5X the listed quantities above, but will exclude the sealing tape.

For the “C” and “V” kits, the supplied 96-well strip plate consists of twelve 8-well strips in a support frame. This enables custom plate configurations.

Description

CST's PathScan® Total IκBα Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total IκBα 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, an IκBα Rabbit Antibody is added to detect the captured IκBα 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 total IκBα protein.

*Antibodies in kit are custom formulations specific to kit.

Specificity/Sensitivity

PathScan® Total IkappaB alpha Sandwich ELISA Kit detects endogenous levels of total IκBα protein. Using PathScan® Phospho-IkappaB alpha (Ser32) 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 this Sandwich ELISA Kit #7360, remains unchanged (Figure 1). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

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).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

PathScan is a registered trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 6

Cell Signaling Technology Logo
Figure 1: Treatment of HeLa cells with TNF-α stimulates phopsphorylation 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.
ELISA Image 1: PathScan<sup>®</sup> Total IkappaB alpha Sandwich ELISA Kit
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 TNFα (10 ng/ml) and lysed after incubation at 37ºC for 5 minutes.
ELISA Image 2: PathScan<sup>®</sup> Total IkappaB alpha Sandwich ELISA Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.