Western blot analysis of extracts from various cell lines using IκBβ (7B4) Mouse mAb.
Western blot analysis of extracts from MCF7 cells, untreated (-) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/ml, 4 hr; +), using IκBβ (7B4) Mouse mAb (upper) and α-Tubulin (11H10) Rabbit mAb #2125 (lower).
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 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.
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 June 2016
Protocol Id: 262
IκBβ (7B4) Mouse mAb recognizes endogenous levels of total IκBβ protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a recombinant protein specific to a carboxy terminal fragment of human IκBβ protein.
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).
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