Western blot analysis of extracts from human cerebellum, mouse brain and rat brain using F3/Contactin (D7D4X) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
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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 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
F3/Contactin (D7D4X) Rabbit mAb recognizes endogenous levels of total F3/contactin protein. The antibody detects a 32 kDa protein of unknown identity in some tissue extracts.Species Reactivity:
Human, Mouse, Rat
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Met774 of human F3/contactin protein.
F3/contactin (CNTN, contactin 1) is a glycosylphosphatidylinositol (GPI)-anchored neural cell adhesion protein belonging to the immunglobulin protein superfamily (1). During early mammalian development, F3/contactin is expressed in granule neuronal progenitor (GNP) cells, where it was shown to promote GNP differentiation, in part by antagonizing sonic hedgehog (SHH)-mediated proliferation (2). Biochemical studies have shown that F3/contactin interacts with the phosphatase PTPRZ on the surface of oligodendrocyte precursor cells, an association that was shown to be essential for oligodendrocyte maturation (3). F3/contactin expression is also abundant in post-mitotic neurons, where its functions as a neural cell adhesion protein have been suggested to play an important role in synaptic plasticity and memory (4). Although primarily associated with neuronal development and function, F3/contactin expression has also been implicated in extra-neuronal tumorigenesis. For example, expression of F3/contactin was detected in both primary prostate tumors, and lymph node and bone metastases, while patient tumor samples with detectable F3/contactin expression were associated with tumor progression and reduced recurrance-free survival (5).
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