Western blot analysis of extracts from HT-29 cells treated with TNF-α (#2169, 20 ng/ml) and calyculin A (#9902, 100 nM) for 10 minutes as indicated, using Phospho-IκBβ (Thr19/Ser23) Antibody (upper) or IκBβ Antibody #9248 (lower).
|MW (kDa)||48 to 50|
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or 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 November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
Phospho-IκBβ (Thr19/Ser23) Antibody detects endogenous levels of human IκBβ only when phosphorylated at threonine 19 and serine 23. This antibody also recognizes phosphorylation at Ser19/Ser23 also reported as the sequence for IκBβ.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues of human IκBβ surrounding Thr19/Ser23. Antibodies are purified by protein A and affinity chromatography.
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).
The regulation of IκBβ and IκBε is similar to that of IκBα. However, the phosphorylation and ubiquitin-mediated degradation of these proteins occurs with much slower kinetics (9). IKK phosphorylation of IκBβ occurs at Ser19 and Ser23, while IκBε can be phosphorylated at Ser18 and Ser22 (10). The human sequence of IκB-β has also been reported to contain a threonine at position 19 suggesting that phosphorylation could be Thr19/Ser23 (11).
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