Overview

  • Product name

  • Description

    Rabbit polyclonal to HUWE1/Mule
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, WB, IP, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Chimpanzee, Gorilla, Orangutan
  • Immunogen

    Synthetic peptide corresponding to Human HUWE1/Mule aa 2250-2300.
    Database link: Q7Z6Z7

  • Positive control

    • Whole cell lysate from 293T cells.
  • General notes

     This product was previously labelled as HUWE1

     

Properties

Applications

Our Abpromise guarantee covers the use of ab70161 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 1 µg/ml.
WB 1/1000 - 1/5000. Detects a band of approximately >460 kDa (predicted molecular weight: 481 kDa).
IP Use at 2-5 µg/mg of lysate.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Target

  • Function

    E3 ubiquitin-protein ligase which mediates ubiquitination and subsequent proteasomal degradation of target proteins. Regulates apoptosis by catalyzing the polyubiquitination and degradation of MCL1. Mediates monoubiquitination of DNA polymerase beta (POLB) at 'Lys-41', 'Lys-61' and 'Lys-81', thereby playing a role in base-excision repair. Also ubiquitinates the p53/TP53 tumor suppressor and core histones including H1, H2A, H2B, H3 and H4. Binds to an upstream initiator-like sequence in the preprodynorphin gene. Regulates neural differentiation and proliferation by catalyzing the polyubiquitination and degradation of MYCN. May regulate abundance of CDC6 after DNA damage by polyubiquitinating and targeting CDC6 to degradation.
  • Tissue specificity

    Weakly expressed in heart, brain and placenta but not in other tissues. Expressed in a number of cell lines, predominantly in those from colorectal carcinomas.
  • Pathway

    Protein modification; protein ubiquitination.
  • Involvement in disease

    Defects in HUWE1 are the cause of mental retardation syndromic X-linked Turner type (MRXST) [MIM:300706]; also known as mental retardation and macrocephaly syndrome. MRXST shows clinical variability. Associated phenotypes include macrocephaly and variable contractures.
    A chromosomal microduplication involving HUWE1 and HSD17B10 is the cause of mental retardation X-linked type 17 (MRX17) [MIM:300705]; also known as mental retardation X-linked type 31 (MRX31). Mental retardation is characterized by significantly sub-average general intellectual functioning associated with impairments in adaptative behavior and manifested during the developmental period. In contrast to syndromic or specific X-linked mental retardation which also present with associated physical, neurological and/or psychiatric manifestations, intellectual deficiency is the only primary symptom of non-syndromic X-linked mental retardation.
  • Sequence similarities

    Belongs to the TOM1/PTR1 family.
    Contains 1 HECT (E6AP-type E3 ubiquitin-protein ligase) domain.
    Contains 1 UBA domain.
    Contains 1 UIM (ubiquitin-interacting motif) repeat.
    Contains 1 WWE domain.
  • Domain

    The HECT domain mediates inhibition of the transcriptional activity of p53.
  • Post-translational
    modifications

    Phosphorylated on tyrosine; phosphorylation is probably required for its ability to inhibit TP53 transactivation.
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization

    Cytoplasm. Nucleus. Mainly expressed in the cytoplasm of most tissues, except in the nucleus of spermatogonia, primary spermatocytes and neuronal cells (By similarity). Predominantly cytosolic or perinuclear in some colorectal carcinoma cells.
  • Information by UniProt
  • Database links

  • Alternative names

    • ARF binding protein 1 antibody
    • ARF BP1 antibody
    • ARF-binding protein 1 antibody
    • ARF-BP1 antibody
    • ARFBP1 antibody
    • BJ-HCC-24 tumor antigen antibody
    • E3 ubiquitin protein ligase HUWE1 antibody
    • E3 ubiquitin-protein ligase HUWE1 antibody
    • HECT antibody
    • HECT domain protein LASU1 antibody
    • HECT UBA and WWE domain containing protein 1 antibody
    • HectH9 antibody
    • Homologous to E6AP carboxyl terminus homologous protein 9 antibody
    • Huwe1 antibody
    • HUWE1_HUMAN antibody
    • Ib772 antibody
    • Large structure of UREB1 antibody
    • LASU1 antibody
    • Mcl 1 ubiquitin ligase E3 antibody
    • Mcl-1 ubiquitin ligase E3 antibody
    • Mule antibody
    • UBA and WWE domain-containing protein 1 antibody
    • Upstream regulatory element-binding protein 1 antibody
    • URE-B1 antibody
    • URE-binding protein 1 antibody
    • UREB1 antibody
    see all

Images

  • All lanes : Anti-HUWE1/Mule antibody (ab70161) at 1 µg/ml

    Lane 1 : 293T whole cell lysate at 50 µg
    Lane 2 : 293T whole cell lysate at 15 µg
    Lane 3 : 293T whole cell lysate at 5 µg

    Predicted band size: 481 kDa
    Observed band size: >460 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 200 kDa, 300 kDa. We are unsure as to the identity of these extra bands.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling HUWE1/Mule with ab70161 at 1/1000 (1µg/ml). Detection: DAB.

  • ICC/IF image of ab70161 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab70161, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • IHC image of ab70161 staining in human normal heart formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with EDTA (pH9, epitope retrieval solution 2) for 20 mins. The section was then incubated with ab70161, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:

See all 6 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293)
Gel Running Conditions
Reduced Denaturing (10%)
Loading amount
50 µg
Specification
HEK293
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jan 29 2014

Answer

Thank you for your email.
I have added a vial of ab70161 in Order 1086585. It wasn't possible to add the vial in the current "dispatch today" order.
I hope this informaiton will be helpful.
Have a good weekend!

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Answer

Thank you for taking time to complete our questionnaire and for contacting us. I am sorry to hear this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality. I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful.
Having reviewed the protocol details, I believe this product should have given satisfactory results. It appears that you may have received a faulty vial.
I apologize for the inconvenience and am pleased to offer you a free of charge replacement, credit note, or refund in compensation.
Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Question

Complain for WB:
1) Abcam catalogue no:
Ab70161
2) a) Abcam order number (not required):
1065472
b) Abcam product lot number:
GR 38197-5 3) Description of the problem (high background, wrong band size, more bands, no band etc):
Background, non-specific bands
4) Antibody storage conditions (temperature/reconstitution etc):
-20 C
5) a)Sample (Species):
Human oral cancer cell lines
b) Type of sample (Cell extract/Nuclear extract/Purified protein/Recombinant protein etc).:Whole cell lysate
6) a) Sample preparation:
Lysis buffer
Fermentas mammalian cell lysis buff
Protease inhibitors
Protease inhibitor cocktail from Fermentas
Phosphatase inhibitors
NaF, Sodium orthovanadate, Beta glycerophosphate
Reducing agent BMEBoiling for ≥5 min?
Exactly for 5 mins
b) Amount of protein loaded:
Protein loaded ug/lane or cells/lane
30μg
7) a) Electrophoresis/Gel conditions:
Reducing or Non Reducing gelNon Reducing gel
Percentage of gel10%
Volts applied80 V
Time applied
b) Transfer or blocking conditions:
Type of membrane
PVDF
Protein transfer verified
By Ponceaue staining
Blocking agent and concentration
Both 5% skimmed Milk and 3% BSA
Blocking time 1 ho
Blocking temperature Room temperature
8) Primary antibody (If more than one was used, describe in “additional notes”):
Concentration or dilution 1:500, 1:750, 1:1000
Diluent buffer Milk or BSA in TBS
Incubation time Overnight
Incubation temperature4 C
Washing: Buffer Used TBST
Number of washes 6 of 10 mins each
9) Secondary antibody:
Species goat
Reacts against rabbit

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Answer

Thank you for your email. I am sorry to hear that you are experiencing problems with this antibody.
I have checked the protocol and images you have kindly sent I however have few following questions:
- The molecular weight of ARFBP1 is ˜400 kda we so recommend using 8% gel.
- You have described that oral cancer cell line was used as negative control; is it true Human oral cancer cell lines was also used as cell line to be tested.
- In the image what does MU stands for.
- The 1/500 dilution seems more promising with 5% milk, have you repeated the experiment using the same conditions.
I will look forward to hearing from you soon.

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