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
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.
LATS1 Antibody detects endogenous levels of total LATS1 protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to amino acids surrounding Ser177 of human LATS1. Antibodies are purified by protein A and peptide affinity chromatography.
LATS1 (Large tumor suppressor 1) is a putative serine/threonine kinase that belongs to the NDR family (1). It is a tumor suppressor that plays a critical role in the maintenance of ploidy. LATS1 localizes to the centrosome and the mitotic spindle and controls G2/M transition by negatively regulating cdc2 kinase activity (2,3). LATS1 also plays a role in the G1 tetraploidy checkpoint via control of p53 expression (4).
LATS1 affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1 (5). LATS1 also binds the phosphorylated form of zyxin, a regulator of actin filament assembly. This interaction promotes localization of zyxin to the mitotic spindle, suggesting a role for actin regulatory proteins during mitosis (6). Decreased expression is associated with breast tumor aggressiveness (7), promoter methylation, and loss of heterozygosity. Mutations perturbing LATS1 have been associated with human sarcomas and ovarian sarcomas (8,9). LATS1 knock out mice develop soft-tissue sarcomas, ovarian stromal cell tumor, and display a high sensitivity to carcinogenic treatments (10).
Human LATS1 exists in a complex similar to the Drosophila Hippo/Salvador/Lats tumor suppressor network, a complex that regulates proliferation and apoptosis to control growth and shape of the fly. The corresponding human complex contains Hippo and Salvador homologs RASSF1A, WW45, and MST2 and may control mitotic exit (11).
Many molecules have been discovered that are involved in the Hippo pathway, but mechanisms governing activation of these proteins and activation of the pathway in general are currently not clear. Phosphorylation of LATS1 and LATS2 is stimulated by association with Mob1, which is itself phosphorylated by Mst1/2 (12,13). Phosphorylation of LATS1/2 is required for activation of YAP, a key mediator of the Hippo signaling pathway (14).
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|9153S||100 µl (10 western blots)||$255.00.0|