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5149
DUSP4/MKP2 (D9A5) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

DUSP4/MKP2 (D9A5) Rabbit mAb #5149

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  1. WB
Western Blotting Image 1: DUSP4/MKP2 (D9A5) Rabbit mAb

Western blot analysis of extracts from various cell lines using DUSP4/MKP2 (D9A5) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

To Purchase # 5149S
Product # Size Price
5149S
100 µl $ 268

Supporting Data

REACTIVITY H Mk
SENSITIVITY Endogenous
MW (kDa) 42
Source/Isotype Rabbit 

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000

Storage

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.

Protocol

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Western Blotting Protocol

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 product webpage for recommended antibody dilution.

A. Solutions and Reagents

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.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Bovine Serum Albumin (BSA): (#9998).
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Biotinylated Protein Ladder Detection Pack: (#7727).
  13. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  15. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  16. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-rabbit IgG, HRP-linked Antibody (#7074 at 1:2000) and anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 10

Specificity / Sensitivity

DUSP4/MKP2 (D9A5) Rabbit mAb recognizes endogenous levels of total DUSP4 protein.

Species Reactivity:

Human, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro168 of human DUSP4 protein.

Background

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).

  1. Camps, M. et al. (2000) FASEB J 14, 6-16.
  2. Theodosiou, A. and Ashworth, A. (2002) Genome Biol 3, REVIEWS3009.
  3. Salojin, K. and Oravecz, T. (2007) J Leukoc Biol 81, 860-9.
  4. Tanoue, T. et al. (2002) J Biol Chem 277, 22942-9.
  5. Dickinson, R.J. and Keyse, S.M. (2006) J Cell Sci 119, 4607-15.
  6. Wu, G.S. (2007) Cancer Metastasis Rev 26, 579-85.
  7. Peng, D.J. et al. (2010) Cell Cycle 9, 4650-5.
  8. Lawan, A. et al. (2011) J Biol Chem 286, 12933-43.
  9. Cagnol, S. and Rivard, N. (2012) Oncogene , .
  10. Chitale, D. et al. (2009) Oncogene 28, 2773-83.
  11. Waha, A. et al. (2010) Cancer Res 70, 1689-99.
  12. Balko, J.M. et al. (2012) Nat Med 18, 1052-9.
  13. Ramesh, S. et al. (2008) EMBO Rep 9, 990-7.
  14. Cornell, T.T. et al. (2010) Infect Immun 78, 2868-76.
  15. Huang, C.Y. et al. (2012) Eur J Immunol 42, 476-88.
  16. Yu, M. et al. (2012) Proc Natl Acad Sci U S A 109, E879-88.

Pathways & Proteins

Explore pathways + proteins related to this product.

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