Western blot analysis of extracts from U-2 OS and MCF7 cells using LIMD1 Antibody.
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
LIMD1 Antibody recognizes endogenous levels of total LIMD1 protein. This antibody cross-reacts with a protein of unknown origin at ~45 kDa.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human LIMD1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
LIM domain-containing protein 1 (LIMD1) is a putative tumor suppressor and adapter/scaffold protein that belongs to the Ajuba family of LIM domain containing proteins. LIM domain containing proteins mediate protein-protein interactions and typically contain a pair of distinct zinc finger domains (1). Research studies indicate that LIMD1 is involved in numerous cellular processes, including inhibition of E2F mediated transcription (2) and negative regulation of the Hippo pathway through influence on YAP phosphorylation state (3,4). Additional studies identify LIMD1 as a hypoxia regulator as it recruits the Von Hippel-Lindau (VHL) protein and the hydroxylase PHD1 to a protein complex that promotes initiation of HIF-1α ubiquitination and degradation (5). Research evidence supporting the role of LIMD1 as a tumor suppressor includes the down regulation of the protein in 80% of lung cancers (6), loss of LIMD1 expression in head and neck cancers (7), and altered subcellular localization in cases of breast cancer (8).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.