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14321
NAE1/APPBP1 (D9I4Z) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

NAE1/APPBP1 (D9I4Z) Rabbit mAb #14321

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  1. WB
Western Blotting Image 1: NAE1/APPBP1 (D9I4Z) Rabbit mAb

Western blot analysis of extracts from various cell lines using NAE1/APPBP1 (D9I4Z) Rabbit mAb.

Western Blotting Image 2: NAE1/APPBP1 (D9I4Z) Rabbit mAb

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human NAE1/APPB1 protein (hNAE1-Myc/DDK; +), using NAE1/APPBP1 (D9I4Z) Rabbit mAb.

To Purchase # 14321S
Product # Size Price
14321S
100 µl N/A

Supporting Data

REACTIVITY H Mk M R
SENSITIVITY Endogenous
MW (kDa) 60
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

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

NAE1/APPBP1 (D9I4Z) Rabbit mAb recognizes endogenous levels of total NAE1/APPBP1 protein. This antibody does not cross-react with E1 activating enzymes for either ubiquitin or other ubiquitin-like proteins.

Species Reactivity:

Human, Monkey, Mouse, Rat

Species predicted to react based on 100% sequence homology:

Bovine, Dog

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala179 of human NAE1/APPBP1 protein.

Background

Similar to ubiquitin, NEDD8 is covalently linked to target proteins through an enzymatic cascade composed of NEDD8-specific E1 (activating)- and E2 (conjugating)-enzymes (1,2). The E2 ligase specific for NEDD8 is Ubc12 (3-5). Ubc12 forms a heterodimeric conjugate with NEDD8 in order to catalyze the transfer of NEDD8 from E1 to lysine side chains of target proteins (1,2). Well known targets of NEDD8 are cullin-based RING E3 ligases. Neddylation of cullin isoforms activates the related ubiquitin E3 complex by promoting its interaction with a cognate ubiquitin-E2 ligase (6-7). Neddylation of Cul-1 complexes containing βTrCP and SKP2 has been shown to be required for controlling the stability of important signaling targets such as IκB, NF-κB, and p27 Kip (8-10), thereby regulating cell cycle progression, signaling cascades, and developmental programming processes (11).

NAE1/APPBP1 (NEDD8-Activating Enzyme 1/Amyloid Beta Precursor Protein Binding Protein 1) exists in a heterodimeric complex with UBA3. This complex functions as an E1 NEDD8-activating enzyme, which utilizes ATP to adenylate the C-terminal glycine of NEDD8 (12-14). Research studies suggest that inhibition of the APPBP1-UBA3 complex may be of therapeutic value for the treatment of human cancers (15,16).

  1. Huang, D.T. et al. (2007) Nature 445, 394-8.
  2. Huang, D.T. et al. (2005) Mol Cell 17, 341-50.
  3. Liakopoulos, D. et al. (1998) EMBO J 17, 2208-14.
  4. Gong, L. and Yeh, E.T. (1999) J Biol Chem 274, 12036-42.
  5. Wada, H. et al. (2000) J Biol Chem 275, 17008-15.
  6. Sakata, E. et al. (2007) Nat Struct Mol Biol 14, 167-8.
  7. Kawakami, T. et al. (2001) EMBO J. 20, 4003-4012.
  8. Podust, V.N. et al. (2000) Proc Natl Acad Sci USA 97, 4579-84.
  9. Wu, K. et al. (2002) J Biol Chem 277, 516-27.
  10. Amir, R.E. et al. (2002) J Biol Chem 277, 23253-9.
  11. Herrmann, J. et al. (2007) Circ Res 100, 1276-91.
  12. Walden, H. et al. (2003) Mol Cell 12, 1427-37.
  13. Walden, H. et al. (2003) Nature 422, 330-4.
  14. Huang, D.T. et al. (2004) Nat Struct Mol Biol 11, 927-35.
  15. Soucy, T.A. et al. (2009) Nature 458, 732-6.
  16. Swords, R.T. et al. (2010) Blood 115, 3796-800.

Pathways & Proteins

Explore pathways + proteins related to this product.

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