Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24 h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb (upper) and IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower).
Western blot analysis of extracts from NIH/3T3 cells, untreated orTNF-α-treated (#2169, 20 ng/ml) for 5 minutes, using Phospho-IκBα (Ser32/36) (5A5) Mouse mAb (upper) or IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) #4814 (lower).
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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
Phospho-IκBα (Ser32/36) (5A5) Mouse mAb detects endogenous levels of IκBα only when phosphorylated at Ser32/36.
Human, Mouse, Rat, Monkey
Bovine, Dog, Pig, Guinea Pig
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser32/36 of human IκBα.
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).
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