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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous 98 Rabbit IgG

Western blot analysis of extracts from various cell lines using UBE3A (D10D3) Rabbit mAb.

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Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a cDNA expression construct encoding transcript variant 3 of human UBE3A (hUBE3A, +), using UBE3A (D10D3) Rabbit mAb.

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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 datasheet or 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. 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 (Premixed Format): (#7720).
  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 (#7720, 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 datasheet) 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 November 2013

protocol id: 10

Western Blot Reprobing Protocol

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.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Wash Buffer: Tris Buffered Saline with Tween® 20 (TBST-10X) (#9997)
  2. Stripping Buffer: To prepare 100 ml, mix 0.76 g Tris base, 2 g SDS and 700 μl β-mercaptoethanol. Bring to 100 ml with deionized H2O. Adjust pH to 6.8 with HCl.

B. Protocol

  1. After film exposure, wash membrane four times for 5 min each in TBST. Best results are obtained if the membrane is not allowed to dry.
  2. Incubate membrane for 30 min at 50°C in stripping buffer (with slight agitation).
  3. Wash membrane six times for 5 min each in TBST.
  4. (Optional) To assure that the original signal is removed, wash membrane twice for 5 min each with 10 ml of TBST. Incubate membrane with LumiGLO® with gentle agitation for 1 min at room temperature. Drain membrane of excess developing solution. Do not let dry. Wrap in plastic wrap and expose to x-ray film.
  5. Wash membrane again four times for 5 min each in TBST.
  6. The membrane is now ready to reuse. Start detection at the "Membrane Blocking and Antibody Incubations" step in the Western Immunoblotting Protocol.

posted June 2005

Product Usage Information

Application Dilutions
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.

Specificity / Sensitivity

UBE3A (D10D3) Rabbit mAb recognizes endogenous levels of total UBE3A protein. Based upon sequence alignment, this antibody is predicted to cross-react with all UBE3A splice variants.


Species Reactivity: Human, Mouse, Rat, Monkey
Species predicted to react based on 100% sequence homology: Bovine, Dog, Horse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human UBE3A protein.

UBE3A, also commonly referred to as E6AP (E6 Associated Protein), is an E3 ubiquitin protein ligase and founding member of the HECT (Homologous to the E6 Carboxyl Terminus) family of E3 ligases (1). UBE3A has been shown to be hijacked by the oncogenic E6 protein of high-risk human papillomaviruses (HPV16 and HPV18) that causes the ubiquitination activity of UBE3A to be inappropriately directed toward several specific cellular proteins, the most notable of which, with respect to carcinogenesis, is p53 (2). Although the DNA-repair enzyme, HHR23A (human homolog A of Rad23), was the first described E6-independent substrate of UBE3A, very few E6-independent targets of UBE3A have been identified. This continues to be an active area of research, particularly because mutations or disruption in expression of UBE3A in the brain are the cause of Angelman syndrome (AS), a severe form of mental retardation (3-6). Although UBE3A is expressed in most human tissues from both parental alleles, it is expressed from the maternal allele in subregions of the brain, with the paternal allele being epigenetically silenced. AS is caused by disruptions in expression of the materal UBE3A allele, generally by large chromosomal deletion, but also by point mutations within the UBE3A coding sequence. This strongly suggests that lack of ubiquitination of one or more UBE3A substrates in neuronal tissue is responsible for the AS phenotype (7). Indeed, a recent study identified several new neuronal substrates of UBE3A including Arc and Ephexin-5 (8). The immediate early gene Arc (activity-regulated cytoskeleton-associated protein) is rapidly upregulated after robust neuronal stimulation and promotes internalization of AMPA-type glutamate receptors (AMPARs), resulting in reduction in synaptic strength. UBE3A ubiquitinates Arc and promotes its degradation by the 26S proteasome, thus preventing AMPAR internalization (8). Disruption in neuronal UBE3A function leads to an increase in Arc expression and a decrease in AMPARs at excitatory synapses, which may contribute to the neurological symptoms of AS.


1.  Huibregtse, J.M. et al. (1995) Proc Natl Acad Sci U S A 92, 5249.

2.  Huibregtse, J.M. et al. (1993) Mol Cell Biol 13, 775-84.

3.  Fang, P. et al. (1999) Hum Mol Genet 8, 129-35.

4.  Jiang, Y. et al. (1999) Am J Hum Genet 65, 1-6.

5.  Jiang, Y.H. et al. (1998) Neuron 21, 799-811.

6.  Kumar, S. et al. (1999) J Biol Chem 274, 18785-92.

7.  Mabb, A.M. et al. (2011) Trends Neurosci 34, 293-303.

8.  Greer, P.L. et al. (2010) Cell 140, 704-16.


Entrez-Gene Id 7337
Swiss-Prot Acc. Q05086


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