Western blot analysis of extracts from HeLa cells, untreated or treated with TNF-α (20 ng/ml) and Calyculin A #9902 (50 nM), using Phospho-IKKγ (Ser376) Antibody #2689 (top) or IKKγ (DA10-12) Mouse mAb (bottom).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised June 2020
Protocol Id: 19
IKKγ (DA10-12) Mouse mAb detects endogenous levels of total IKKγ protein.
Monoclonal antibody is produced by immunizing animals with full length human GST-IKKγ protein.
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 and IKKγ serves as the regulatory subunit (8,9). Activation of IKK depends upon phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (Ser176 and Ser180 in IKKα), which causes conformational changes, resulting in kinase activation (10-13).
Activation of the NF-κB pathway by the T-cell lymphotrophic virus Tax protein or by TNF-α treatment leads to IKKβ-dependent phosphorylation of human IKKγ, primarily at Ser376 (14). In mice, mutation of the orthologous residue (Ser369) to alanine leads to enhanced IKKγ-mediated stimlulation of IKKβ kinase activity (15).
Explore pathways + proteins related to this product.
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