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Neuroscience Neurology process Neurodegenerative disease Huntington's disease
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

  • Datasheet
  • SDS
  • Certificate of Compliance
Submit a review Submit a question References (4)

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Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Flow Cytometry - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
  • Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EP867Y] to Huntingtin - BSA and Azide free
  • Suitable for: WB, IHC-P, Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human

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Overview

  • Product name

    Anti-Huntingtin antibody [EP867Y] - BSA and Azide free
    See all Huntingtin primary antibodies
  • Description

    Rabbit monoclonal [EP867Y] to Huntingtin - BSA and Azide free
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
    Unsuitable for: IP
  • Species reactivity

    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Immunogen

    This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: SH-SY5Y, HAP1, and HeLa cell lysates. ICC/IF: SKNSH cells. Flow Cyt: SH-SY5Y cells. IHC-P: Human brain tissue.
  • General notes

    ab225573 is the carrier-free version of ab45169. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    ab225573 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C. Do Not Freeze.
  • Storage buffer

    pH: 7.20
    Constituent: PBS
  • Carrier free

    Yes
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP867Y
  • Isotype

    IgG
  • Research areas

    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Huntington's disease

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Huntingtin antibody [EP867Y] (ab206048)
    • Alexa Fluor® 647 Anti-Huntingtin antibody [EP867Y] (ab206049)
    • APC Anti-Huntingtin antibody [EP867Y] (ab224969)
    • PE Anti-Huntingtin antibody [EP867Y] (ab224970)
    • Anti-Huntingtin antibody [EP867Y] (ab45169)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (ab96899)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
  • KO cell lysates

    • Human HTT (Huntingtin) knockout HeLa cell lysate (ab256946)
  • Recombinant Protein

    • Recombinant Human Huntingtin protein (Tagged) (ab112300)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab225573 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
WB
Use at an assay dependent concentration. Predicted molecular weight: 348 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt
Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 348 kDa.
IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt
Use at an assay dependent concentration.

ab199376 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for IP.
  • Target

    • Function

      May play a role in microtubule-mediated transport or vesicle function.
    • Tissue specificity

      Expressed in the brain cortex (at protein level). Widely expressed with the highest level of expression in the brain (nerve fibers, varicosities, and nerve endings). In the brain, the regions where it can be mainly found are the cerebellar cortex, the neocortex, the striatum, and the hippocampal formation.
    • Involvement in disease

      Defects in HTT are the cause of Huntington disease (HD) [MIM:143100]. HD is an autosomal dominant neurodegenerative disorder characterized by involuntary movements (chorea), general motor impairment, psychiatric disorders and dementia. Onset of the disease occurs usually in the third or fourth decade of life and symptoms progressively worsen leading to death in 10 to 20 years. Onset and clinical course depend on the degree of poly-Gln repeat expansion, longer expansions resulting in earlier onset and more severe clinical manifestations. HD affects 1 in 10,000 individuals of European origin. Neuropathology of Huntington disease displays a distinctive pattern with loss of neurons, especially in the caudate and putamen (striatum).
    • Sequence similarities

      Belongs to the huntingtin family.
      Contains 10 HEAT repeats.
    • Domain

      The N-terminal Gln-rich and Pro-rich domain has great conformational flexibility and is likely to exist in a fluctuating equilibrium of alpha-helical, random coil, and extended conformations.
    • Post-translational
      modifications

      Cleaved by apopain downstream of the polyglutamine stretch. The resulting N-terminal fragment is cytotoxic and provokes apoptosis.
      Forms with expanded polyglutamine expansion are specifically ubiquitinated by SYVN1, which promotes their proteasomal degradation.
    • Cellular localization

      Cytoplasm. Nucleus. The mutant Huntingtin protein colocalizes with AKAP8L in the nuclear matrix of Huntington's disease neurons.
    • Target information above from: UniProt accession P42858 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      Information by UniProt
    • Database links

      • Entrez Gene: 3064 Human
      • Entrez Gene: 15194 Mouse
      • Entrez Gene: 29424 Rat
      • Omim: 143100 Human
      • Omim: 613004 Human
      • SwissProt: P42858 Human
      • SwissProt: P42859 Mouse
      • SwissProt: P51111 Rat
      • Unigene: 518450 Human
      • Unigene: 209071 Mouse
      • Unigene: 482929 Mouse
      • Unigene: 11193 Rat
      see all
    • Alternative names

      • AI256365 antibody
      • C430023I11Rik antibody
      • HD antibody
      • HD protein antibody
      • HD_HUMAN antibody
      • HDH antibody
      • HTT antibody
      • Huntingtin antibody
      • HUNTINGTON CHOREA antibody
      • Huntington disease protein antibody
      • Huntington's disease protein homolog antibody
      • IT 15 antibody
      • IT15 antibody
      • OTTMUSP00000026909 antibody
      • ZHD antibody
      see all

    Images

    • Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      All lanes : Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : HTT knockout HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 348 kDa
      Observed band size: 348 kDa



      This data was developed using the same antibody clone in a different buffer formulation (ab45169).

        Lanes 1- 2: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

       ab45169 was shown to react with Huntingtin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265976 (knockout cell lysate ab256946) was used. Wild-type HeLa and HTT knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab45169 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

      Ab45169 staining human Huntingtin in human brain tissue by immunohistochemistry using paraffin embedded tissue.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

      ICC/IF image of ab45169 stained SKNSH cells. The cells were 100% methanol fixed (5 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 (ab45169, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).

    • Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Western blot - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      All lanes : Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : Huntingtin knockout HAP1 whole cell lysate
      Lane 3 : SH-SY-5Y whole cell lysate
      Lane 4 : HeLa whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 348 kDa



      This WB data was generated using the same anti-Huntingtin antibody clone [EP867Y] in a different buffer formulation (cat# ab45169).

      Lanes 1 - 4: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - loading control, ab18058, observed at 130 kDa.

      ab45169 was shown to recognize Huntingtin when Huntingtin knockout samples were used, along with additional cross-reactive bands. Wild-type and Huntingtin knockout samples were subjected to SDS-PAGE. ab45169 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C both at 1/10000 dilution. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

    • Flow Cytometry - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Flow Cytometry - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

      Overlay histogram showing SH-SY5Y cells stained with ab45169 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45169, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) ( 1µg/1x10^6 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

      This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).

    • Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
      Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (4)

    Publishing research using ab225573? Please let us know so that we can cite the reference in this datasheet.

    ab225573 has been referenced in 4 publications.

    • Lin CL  et al. Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington's disease via suppressing oxidative stress, inflammation, and apoptosis. Clin Interv Aging 10:1173-87 (2015). IHC-P ; Mouse . PubMed: 26229452
    • Czeredys M  et al. Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease. Front Mol Neurosci 6:42 (2013). WB ; Human . PubMed: 24324398
    • Clemen CS  et al. Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases. Brain 133:2920-41 (2010). PubMed: 20833645
    • Cho KJ  et al. Inhibition of apoptosis signal-regulating kinase 1 reduces endoplasmic reticulum stress and nuclear huntingtin fragments in a mouse model of Huntington disease. Neuroscience 163:1128-34 (2009). PubMed: 19646509

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