Western blot analysis of extracts from various cell types using MCF2/Dbl Antibody.
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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 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
MCF2/Dbl Antibody recognizes endogenous levels of total MCF2/Dbl protein. Based on amino acid sequence homology, the antibody is expected to recognize splice variants 1-4 of human MCF2/Dbl. The antibody is not expected to recognize the onco-Dbl protein formed through amino-terminal truncation.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the amino terminus of human MCF2/Dbl. Antibodies are purified using protein A and peptide affinity chromatography.
The MCF2/Dbl proto-oncogene product is the founding member of the Dbl family of Rho guanine nucleotide exchange factors (GEFs) that are characterized by their Dbl homology (DH) domain (1). GEFs stimulate the formation of the active, GTP-bound form of small GTPases such as Rho, Rac and Cdc42, signaling to various downstream molecules and regulating diverse cell functions. While the overexpressed, full-length Dbl gene has transforming activity (2), mutations resulting in truncated Dbl cause the protein to become highly oncogenic. This truncated form of Dbl, which lacks the amino-terminal 497 amino acids, has constitutive GEF activity (3) and is more stable than the full-length variant (4), allowing for increased signaling to downstream effector molecules.
Dbl interacts with ezrin, a member of the ezrin/radixin/moesin (ERM) family of proteins that links the plasma membrane to the actin cytoskeleton. Dbl interacts with ezrin in lipid microdomains, which leads to Cdc42 activation and the regulation of processes such as filopodia formation and cell polarity (5,6). Dbl localization and biological activities are regulated in part by phosphatidylinositol 3-kinase (PI3K) (7). Dbl is also involved in cell survival and inhibits apoptosis through induction of Akt phosphorylation at Thr308 (8).
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