Western blot analysis of extracts from various tissue using Contactin-2 (D4M7G) 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 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
Contactin-2 (D4M7G) Rabbit mAb recognizes endogenous levels of total contactin-2 protein.Species Reactivity:
Mouse, RatSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val375 of human contactin-2 protein.
Myelinated axons contain un-myelinated gaps called nodes of Ranvier. These regularly spaced gaps are critical for the proper propagation and rapid conduction of nerve impulses in the central and peripheral nervous system (1). The structure and organization of the nodes of Ranvier is dictated by interaction between the axon and glial cells (2). Voltage-gated sodium channels concentrated at the nodes and potassium channels clustered at the paranodes are responsible for propagation of the action potentials (3,4). Other proteins that contribute to the architecture and function of the nodes of Ranvier include βIV spectrin (5), ankyrin-G (6), and the L1 cell adhesion molecules, neurofascin and NrCAM (7,8).
Contactin-2 (CNTN2, TAG-1) is a glycosyl-phosphatidyl-inositol-anchored cell adhesion protein that is expressed at the juxtaparanodal region of the nodes of Ranvier by oligodendrocytes, Schwann cells, and axons (9,10). Contactin-2 plays an important role in the proper organization of the juxtaparanodes through interaction with Caspr2 and the recruitment of the Kv1.1 and Kv1.2 potassium channel subunits (11). Research studies indicate that contactin-2 is a substrate of β-secretase 1 (12,13). A deletion mutation in the corresponding CNTN2 gene results in familial adult myoclonic epilepsy-5, which is characterized by seizures, involuntary myoclonic muscle twitches, and mild intellectual disability (14).
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