Western blot analysis of extracts from A431, MCF7, HeLa, C6, NIH/3T3 and C2C12 cells, using Erk3 Antibody.
|REACTIVITY||H M R Mk|
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
Erk3 Antibody detects endogenous levels of total Erk3 protein. The antibody does not cross-react with other other Erk family members.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu410 of human Erk3. Antibodies are purified by protein A and peptide affinity chromatography.
Erk3, also known as MAPK6 or p97 MAPK, is almost 50% identical to Erk1/2 at the kinase domain located in its amino-terminal region (1). However, Erk3 is distinguished from other MAP kinases in that it lacks the conserved TXY motif in its activation loop, possessing instead an SEG motif (1,2). Phosphorylation at Ser189 in the SEG motif has been reported (2,3). With limited information about its upstream kinases and downstream substrates, the significance of this phosphorylation remains to be elucidated (3,4). Erk3 is an inherently unstable protein, rapidly degraded through amino-terminal ubiquitination and proteasome degradation (3,5). A site-specific cleavage, depending on a short stretch of acidic residues of Erk3, might regulate its translocation from the Golgi/ERGIC to the nucleus during the cell cycle (6). Accumulating evidence suggests that Erk3 is involved in cell differentiation (1,3,6).
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