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MetAP2 (D3I1H) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

MetAP2 (D3I1H) Rabbit mAb #12547

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  1. WB
Western Blotting Image 1 - MetAP2 (D3I1H) Rabbit mAb

Western blot analysis of extracts from various cell lines using MetAP2 (D3I1H) Rabbit mAb.

Western Blotting Image 2 - MetAP2 (D3I1H) Rabbit mAb

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing DDK-tagged full-length human MetAP2 (hMetAP2-DDK; +), using MetAP2 (D3I1H) Rabbit mAb.

To Purchase # 12547S
Product # Size Price
100 µl $ 268

Supporting Data

MW (kDa) 63
Source/Isotype Rabbit IgG

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


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.



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Western Blotting Protocol

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 product webpage for recommended antibody dilution.

A. Solutions and Reagents

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. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-rabbit IgG, HRP-linked Antibody (#7074).
  15. 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, 10 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: 263

Specificity / Sensitivity

MetAP2 (D3I1H) Rabbit mAb recognizes endogenous levels of total MetAP2 protein. Based upon sequence alignment, this antibody is not predicted to cross-react with MetAP1.

Species Reactivity:

Human, Mouse, Rat, Monkey

Species predicted to react based on 100% sequence homology:

Hamster, Dog, Pig, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly390 of human MetAP2 protein.


Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67/methionine aminopeptidase 2 (MetAP2) is one of the three known MetAPs responsible for the co-translational processing of the N-terminal initiator methionine from nascent proteins in cells. MetAP2 regulates the rates of global protein synthesis by controlling the levels of eIF2α phosphorylation (1). MetAP2 has also been shown to bind Erk1/2 to inhibit their activation and activity, thus connecting the protein synthesis machinery with the cell signaling pathway mediated by Erk1/2 MAP kinases (2-4). Although MetAP2 is characterized as having aminopeptidase activity that removes the N-terminal methionine from nascent peptides in vitro, mounting evidence suggests that MetAP2 has no methionine aminopeptidase activity. Rather, MetAP2 possesses auto-proteolytic activity that can be inhibited by several small molecule inhibitors including anti-angiogenic drugs, fumagillin and its derivatives (5). It has also been demonstrated that O-GlcNAcylation of MetAP2 plays a major role in its stability, eIF2α binding, and maintenance of eIF2α phosphorylation (6).

MetAP2 knockout mice show embryonic lethality, suggesting its role in embryonic development and survival at the initiation of gastrulation (7). It is likely that lowering the levels of MetAP2 in mammalian cells causes cell growth inhibition and leads to apoptosis due to the high levels of eIF2α phosphorylation that inhibits global protein synthesis (8). During pathological or various stress conditions, MetAP2 dissociates from eIF2 subunits possibly due to its deglycosylation-induced autoproteolytic cleavage. As a result, eIF2α becomes hyperphosphorylated and global protein synthesis is inhibited. eIF2 complex-dissociated MetAP2 also displays a higher affinity toward Erk1/2, which results in the blockade of Erk1/2 activity. Thus, MetAP2 mediates cooperation between cell signaling and protein synthesis machinery to regulate cell growth and proliferation during physiological and pathological conditions (9). Research studies have shown higher expression of MetAP2 in human cancers, supporting the contention that MetAP2 plays a role in oncogenesis. For example, investigators have reported high MetAP2 expression in follicular lymphomas, large B-cell lymphomas, and Burkitt's lymphomas (10). Elevated expression of MetAP2 has also been reported in human colorectal adenocarcinomas (11).

  1. Datta, B. (2000) Biochimie 82, 95-107.
  2. Datta, B. et al. (2004) Arch Biochem Biophys 427, 68-78.
  3. Datta, B. et al. (2004) Biochemistry 43, 14821-31.
  4. Datta, B. et al. (2005) Exp Cell Res 303, 174-82.
  5. Bradshaw, R.A. and Yi, E. (2002) Essays Biochem 38, 65-78.
  6. Datta, B. et al. (1999) Exp Cell Res 250, 223-30.
  7. Yeh, J.R. et al. (2006) Proc Natl Acad Sci U S A 103, 10379-84.
  8. Datta, B. and Datta, R. (1999) Exp Cell Res 246, 376-83.
  9. Ghosh, A. et al. (2006) Exp Cell Res 312, 3184-203.
  10. Kanno, T. et al. (2002) Lab Invest 82, 893-901.
  11. Selvakumar, P. et al. (2004) Clin Cancer Res 10, 2771-5.

Pathways & Proteins

Explore pathways + proteins related to this product.

For Research Use Only. Not For Use In Diagnostic Procedures.
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