Western blot analysis of extracts from various cell lines using DUSP4/MKP2 (D9A5) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (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 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
DUSP4/MKP2 (D9A5) Rabbit mAb recognizes endogenous levels of total DUSP4 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro168 of human DUSP4 protein.
MAP kinases are inactivated by dual-specificity protein phosphatases (DUSPs) that differ in their substrate specificity, tissue distribution, inducibility by extracellular stimuli, and cellular localization. DUSPs, also known as MAPK phosphatases (MKP), specifically dephosphorylate both threonine and tyrosine residues in MAPK P-loops and have been shown to play important roles in regulating the function of the MAPK family (1,2). At least 13 members of the family (DUSP1-10, DUSP14, DUSP16, and DUSP22) display unique substrate specificities for various MAP kinases (3). MAPK phosphatases typically contain an amino-terminal rhodanese-fold responsible for DUSP docking to MAPK family members and a carboxy-terminal catalytic domain (4). These phosphatases can play important roles in development, immune system function, stress responses, and metabolic homeostasis (5). In addition, research studies have implicated DUSPs in the development of cancer and the response of cancer cells to chemotherapy (6).
DUSP4 (MKP2, hVH2) is a nuclear dual-specificity phosphatase that is a negative regulator of Erk1/2 signaling by dephosphorylating and inactivating Erk1/2 in response to growth factors (7,8). Treatment with mitogen or expression of activating mutations of Ras (G12V) or Raf (V600E) promote increased expression of DUSP4 and a coincident decrease in phospho-Erk in the nucleus (9). In contrast, numerous studies have detected decreased expression of DUSP4 in a variety of tumor types, resulting in increased signaling via the Ras/Erk pathway, enhanced tumor growth, and decreased drug sensitivity (10-12). DUSP4/MKP2 also plays an important role in regulating the immune system where it has been implicated in regulating T and B cell proliferation and apoptosis, and adaptive and inflammatory responses (13-16).
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