|H M R Mk||Endogenous||40||Rabbit IgG|
Western blot analysis of extracts from HeLa cells, untreated or treated with human TNF-α (#8902, 20 ng/ml) for 5 minutes, using Phospho-IκBα (Ser32) (14D4) Rabbit mAb (Biotinylated).Learn more about how we get our images
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.
Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP will also visualize the biotinylated markers.
* 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: 266
Supplied in 136 mM NaCl, 2.6 mM KCI, 12 mM sodium phosphate (pH 7.4) dibasic, 2 mg/ml BSA, and 50% glycerol. Store at –20°C. Do not aliquot the antibodies.
Phospho-IκBα (Ser32) (14D4) Rabbit mAb (Biotinylated) detects endogenous levels of IκBα only when phosphorylated at Ser32.
Human, Mouse, Rat, Monkey
Chicken, Bovine, Dog, Pig, Guinea Pig
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser32 of human IκBα.
This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated Phospho-IκBα (Ser32) (14D4) Rabbit mAb #2859.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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|5209S||100 µl (10 western blots)||$299.00.0|