Western blot analysis of extracts from wild type (WT), FE65 knock-out (65KO), FE65L1 knock-out (L1KO) and FE65/FE65L1 double knock-out (DKO) mouse brain lysates, using FE65 Antibody. (Kindly provided by Dr. Suzanne Guénette, MassGeneral Institute for Neurodegenerative Disease, Charlestown, Massachusetts).
Western blot analysis of extracts from mouse and rat brain, using FE65 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
FE65 Antibody detects endogenous levels of FE65. It does not cross-react with FE65L1 or FE65L2.Species Reactivity:
Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues of human FE65. Antibodies are purified by protein A and peptide affinity chromatography.
FE65, FE65L1 and FE65L2 are members of the FE65 protein family. FE65 is an adaptor protein with protein-protein interaction domains including a WW domain followed by two phosphotyrosine interaction domains (PID1 and PID2) (1). Amyloid beta precursor protein (APP) binds to PID2 and undergoes sequential cleavage. First alpha-/beta secretases cleave and release the ectodomain into the extracellular environment. Subsequent processing by the gamma-secretase complex results in the APP intracellular domain (AICD) and the beta-amyloid peptides. The latter A-beta fragments form the main components of amyloid plaques in patients with Alzheimer's disease (2). FE65 family members can regulate APP processing, resulting in elevated levels of A-beta (3). Double knock-out mice of FE65 and FE65L1 display a phenotype that occurs in animals lacking APP family members, supporting a functional interaction between FE65 and APP (4).
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