Cell Signaling Technology

Product Pathways - NF-kappaB Signaling

IKKε Kinase #7553

Cell Signaling Technology offers a full line of protein kinases, substrates, antibody detection reagents and HTScan® kits. Browse our "Reagents for High-Throughput Screening" product listing or contact us at drugdiscovery@cellsignal.com.

Description

Purified recombinant full length human IKKepsilon kinase, supplied as a GST fusion protein.

Source / Purification

The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing full length human IKKε (Met1-Val716) (GenBank Accession No. NM_014002) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.

Gel Staining

Gel Staining

Figure 1. The purity of the GST-IKKε fusion protein was analyzed using SDS/PAGE followed by anti-GST Western blot (A) or Coomassie stain (B).

Kinase Assay - Radiometric

Kinase Assay - Radiometric

Figure 2. IKKε kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 1 µM ATP , 2.5 µg/50 µl PEG20,000, Substrate: Casein, 10 µg/50 µl and Recombinant IKKepsilon: 100 ng/50 µl.

Kinase Assay - DELFIA

Kinase Assay - DELFIA

Figure 3. Dose dependence curve of IKKε kinase activity: DELFIA® data generated using Phospho-(Ser/Thr) Phe Antibody #9631 to detect phosphorylation of substrate peptide #1134 by IKKepsilon kinase. In a 50 µl reaction, increasing amounts of IKKepsilon and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)


Quality Control

The theoretical molecular weight of the GST-IKKepsilon fusion protein is 110 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain and Western blot [Fig.1]. IKKepsilon kinase activity was determined using a radiometric assay [Fig.2]. A kinase dose dependency assay was performed to measure IKKepsilon activity using HTScan® IKKepsilon Kinase Assay Kit #7556 [Fig.3].

Background

The NF-κB/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins (1-3). Most agents that activate NF-κB do so through a common pathway based on phosphorylation-induced, proteasome-mediated degradation of IκB (3-7). The key regulatory step in this pathway involves activation of a high molecular weight IκB kinase (IKK) complex, whose catalysis is generally carried out by three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase. IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends on phosphorylation; Ser177 and Ser181 in the activation loop of IKKβ (176 and 180 in IKKα) are the specific sites whose phosphorylation causes conformational changes resulting in kinase activation (10-13).

Recently, two homologs of IKKα and IKKβ have been described, called IKKε (also known as IKK-i) and TBK-1 (also known as T2K or NAK), and activation of either of these kinases results in NF-κB activation. IKKε contains the kinase domain in its amino-terminus, which shares 30% identity to that of IKKα or IKKβ. IKKε is expressed mainly in immune cells, and may play a special role in the immune response (14-18).

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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