Immunoprecipitation of Erk5 from NIH/3T3 cells, untreated or PDGF-treated, using Erk5 Antibody #3372, followed by Western blot with Phospho-Erk5 (Thr218/Tyr220) Antibody #3371, or Erk5 Antibody #3372.
|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
Phospho-Erk5 (Thr218/Tyr220) Antibody detects immunoprecipitated or transfected levels of Erk5 phosphorylated at threonine 218 and tyrosine 220. This antibody cross-reacts with phosphorylated Erk1 and Erk2. It does not cross-react with phosphorylated p38 MAPK or SAPK/JNK.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr218/Tyr220 of human Erk5. Antibodies are purified by protein A and peptide affinity chromatography.
Erk5 (Mitogen-activated protein kinase 7, Big mitogen-activated protein kinase 1) is a member of the MAPK superfamily implicated in the regulation numerous cellular processes including proliferation, differentiation, and survival (1-4). Like other MAPK family members, Erk5 contains a canonical activation loop TEY motif (Thr218/Tyr220) that is specifically phosphorylated by MAP2K5 (MEK5) in a growth-factor-dependent, Ras-independent mechanism (5-7). For example, EGF stimulation promotes Erk5 phosphorylation that induces its translocation to the nucleus where it phosphorylates MEF2C and other transcriptional targets (5,6). Erk5 is also activated in response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic progenitor cells where it promotes survival and proliferation (7). In neuronal cells, Erk5 is required for NGF-induced neurite outgrowth, neuronal homeostasis, and survival (8,9). Erk5 is thought to play a role in blood vessel integrity via maintenance of endothelial cell migration and barrier function (10-12). Although broadly expressed, research studies have shown that mice lacking erk5 display numerous cardiac defects, suggesting Erk5 plays a critical role in vascular development and homeostasis (1,2).
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