|H M R||Endogenous||100||Rabbit IgG|
Western blot analysis of extracts from various cell lines using α-Actinin 4 (D7U5A) Rabbit mAb.Learn more about how we get our images
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human α-actinin proteins (+), using α-Actinin 4 (D7U5A) Rabbit mAb (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).Learn more about how we get our images
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
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.
α-Actinin 4 (D7U5A) Rabbit mAb recognizes endogenous levels of total α-actinin 4 protein. This antibody does not cross-react with other α-actinin proteins.
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala6 of human α-actinin 4 protein.
α-Actinin belongs to the spectrin family of cytoskeletal proteins. It was first recognized as an actin cross-linking protein, forming an antiparallel homodimer with an actin binding head at the amino terminus of each monomer. The α-actinin protein interacts with a large number of proteins involved in signaling to the cytoskeleton, including those involved in cellular adhesion, migration, and immune cell targeting (1). The interaction of α-actinin with intercellular adhesion molecule-5 (ICAM-5) helps to promote neurite outgrowth (2). In osteoblasts, interaction of α-actinin with integrins stabilizes focal adhesions and may protect cells from apoptosis (3). The cytoskeletal α-actinin isoforms 1 and 4 (ACTN1, ACTN4) are non-muscle proteins that are present in stress fibers, sites of adhesion and intercellular contacts, filopodia, and lamellipodia. The muscle isoforms 2 and 3 (ACTN2, ACTN3) localize to the Z-discs of striated muscle and to dense bodies and plaques in smooth muscle (1).
The ubiquitously expressed α-actinin 4 (ACTN4) protein typically localizes to the cytoskeleton but extracellular stimuli will prompt nuclear translocation in some cells (4). Research studies suggest that ACTN4 can mediate cell signaling and regulate gene expression as a transcriptional coactivator (5,6). Increased expression of ACTN4 protein and amplification of the ACTN4 gene are seen in many cancers and correlates with poor prognosis and metastasis (7). Mutations in the corresponding ACTN4 gene are responsible for focal segmental glomerulosclerosis (FSGS1), a severe renal disorder characterized by reduced kidney function, proteinuria, and progressive kidney failure (8).
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|15145S||100 µl (10 western blots)|
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