Western blot analysis of extracts from HeLa, NIH/3T3, C6 and COS cells using MLK3 Antibody.
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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 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: 10
MLK3 Antibody detects endogenous levels of total MLK3 protein. This antibody does not cross-react with MLK1, MLK2 and other mixed lineage kinases.
Human, Mouse, Rat, Hamster, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human MLK3. Antibodies are purified by protein A and peptide affinity chromatography.
Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase that has an amino-terminal SH3 domain followed by the kinase domain and two leucine zippers, a cdc42/Rac1 binding (CRIB) domain and several other domains/motifs at the carboxy-terminal region. CRIB triggers the dimerization of MLK3 via its tandem leucine zippers, followed by the intramolecular phosphorylation and subsequent activation of MLK3 (1,2). Autophosphorylation of Thr277 and Ser281 is essential for MLK3 kinase activity (3). Ser281 is also phosphorylated by HPK in an in vitro kinase assay (3). MLK3 functions as a MAPKKK of the SAPK/JNK stress pathway by directly phosphorylating SEK1/MKK4 and MKK7, although it is controversial whether MLK3 is involved in p38 stress pathway activation (1,4). MLK3 also functions as an IκB kinase and mediates the activation of the transcriptional factor NF-κB stimulated by CD3/CD28, suggesting a role for MLK3 in immune and inflammatory responses (5).
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