Western blot analysis of extracts from HeLa and C6 cells, untreated or UV-treated, using Phospho-SEK1/MKK4 (Ser257/Thr261) Antibody (upper) or SEK1/MKK4 Antibody #9152 (lower).
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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-SEK1/MKK4 (Ser257/Thr261) Antibody detects endogenous levels of SEK1/MKK4 phosphorylated at serine 257/threonine 261 . This antibody does not cross-react with the corresponding phosphorylated residues of MEK1, MEK2 or MKK3. It will also react with SEK1/MKK4 singly phosphorylated at Thr261.Species Reactivity:
Human, Mouse, Rat, D. melanogasterSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser257/Thr261 of human SEK1/MKK4. Antibodies are purified by protein A and peptide affinity chromatography.
SAPK/Erk kinase (SEK1), also known as MKK4 or Jun kinase kinase (JNKK), activates the MAP kinase homologues SAPK and JNK in response to various cellular stresses and inflammatory cytokines (1-3). Activation of SEK1 occurs through MEKK phosphorylation of serine and threonine residues at positions 257 and 261, respectively. Like MEK, SEK is a dual-specificity protein kinase that phosphorylates SAPK/JNK at a conserved T*PY* site in its activation loop (4). Phosphorylation by Akt at Ser80 inhibits SEK1 and suppresses stress-activated signal transduction (5).
Explore pathways + proteins related to this product.
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