Western blot analysis of extracts from COS cells, untransfected (lane 1), transfected with Wild-type LIMK1 (lanes 2 and 3) or with LIMK1 T508A mutant (lanes 4 and 5), using Phospho-LIMK1 (Thr508)/LIMK2 (Thr505) Antibody (top), LIMK1 Antibody #3842 (middle) or HA-Tag (262K) mAb #2362 (bottom). Cells were either untreated (lanes 1, 2 and 4) or treated with PMA (lanes 3 and 5). (Triple HA-tagged LIMK1 plasmids kindly provided by Dr. K. Mizuno, Biological Institute, Tohoku University, Japan.)
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
Phospho-LIMK1 (Thr508)/LIMK2 (Thr505) Antibody detects transfected levels of LIMK1 and LIMK2 only when phosphorylated at threonine 508 or 505.
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr508 of human LIMK1. Antibodies are purified by protein A and peptide affinity chromatography.
LIM kinases (LIMK1 and LIMK2) are serine/threonine kinases that have two zinc finger motifs, known as LIM motifs, in their amino-terminal regulatory domains (1). LIM kinases are involved in actin cytoskeletal regulation downstream of Rho-family GTPases, PAKs, and ROCK (2,3). PAK1 and ROCK phosphorylate LIMK1 or LIMK2 at the conserved Thr508 or Thr505 residues in the activation loop, increasing LIMK activity (3-5). Activated LIM kinases inhibit the actin depolymerization activity of cofilin by phosphorylation at the amino-terminal Ser3 residue of cofilin (6,7).
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