Western blot analysis of extracts from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human Lefty1 (hLefty1-Myc/DDK; +), using Lefty1 (D7E3G) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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
Lefty1 (D7E3G) Rabbit mAb recognizes transfected levels of total lefty1 protein.Species Reactivity:
HumanSpecies predicted to react based on 100% sequence homology:
Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro108 of human lefty1 protein.
Lefty proteins are members of the TGF-β family of cell signaling molecules that are involved in growth and development (1,2). Named for their role in left-right axis determination and their exclusive expression on the left side of the developing mouse embryo, lefty1 and lefty2 contain a cysteine-knot motif that is characteristic of TGF-β related proteins, but lack an alpha-helix and a cysteine residue critical for ligand dimerization (3). Early in vertebrate embryogenesis, lefty represses TGF-β signaling by inhibiting the phosphorylation of Smad2 following activation of the TGF-β receptor (4). Down-regulated very early upon differentiation (5), lefty proteins act as extracellular antagonists of the signaling pathway for Nodal, a TGF-β ligand critical for left-right patterning and formation of the mesoderm and endoderm (6). Similar to other members of the TGF-β superfamily, lefty proproteins undergo cleavage to release a bioactive protein (7). The biologically active 42 kDa lefty precursor and the 28 kDa polypeptide have been shown to induce MAPK activity (7).
Explore pathways + proteins related to this product.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.