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    products/cell-lines/human-htt-huntingtin-knockout-hela-cell-line-ab265976.pdf

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Human HTT (Huntingtin) knockout HeLa cell line (ab265976)

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Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
  • Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
  • Sanger Sequencing - Human HTT knockout HeLa cell line (ab265976)
  • Cell Culture - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)

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Overview

  • Product name

    Human HTT (Huntingtin) knockout HeLa cell line
  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 21
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing
  • Tested applications

    Suitable for: WBmore details
  • Biosafety level

    2
  • General notes

    Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

    Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

    Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

    Culture medium: DMEM (High Glucose) + 10% FBS

    Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

    1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
    2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
    3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
    4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.

    Subculture guidelines:

    • All seeding densities should be based on cell counts gained by established methods.
    • A guide seeding density of 2x104 cells/cm2 is recommended.
    • A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
    • Cells should be passaged when they have achieved 80-90% confluence.

    This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.

Properties

  • Number of cells

    1 x 106 cells/vial, 1 mL
  • Viability

    ~80%
  • Adherent /Suspension

    Adherent
  • Tissue

    Cervix
  • Cell type

    epithelial
  • Disease

    Adenocarcinoma
  • Gender

    Female
  • STR Analysis

    Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
  • Antibiotic resistance

    Puromycin 1.00µg/ml
  • Mycoplasma free

    Yes
  • Storage instructions

    Shipped on Dry Ice. Store in liquid nitrogen.
  • Storage buffer

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Purity

    Immunogen affinity purified
  • Research areas

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

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

Associated products

  • KO cell lysates

    • Human HTT (Huntingtin) knockout HeLa cell lysate (ab256946)
  • Related Products

    • Anti-Huntingtin antibody [EPR5526] (ab109115)
    • Anti-Huntingtin antibody [EPR5526] - BSA and Azide free (ab209668)
    • Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)
    • Anti-Huntingtin antibody [EP867Y] (ab45169)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab265976 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.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 348 kDa.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

Images

  • Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
    Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
    All lanes : Anti-Huntingtin antibody [EPR5526] (ab109115) 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



    Lanes 1- 2: Merged signal (red and green). Green - ab109115 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

     ab109115 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. ab109115 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.

  • Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
    Western blot - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
    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



    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.

  • Sanger Sequencing - Human HTT knockout HeLa cell line (ab265976)
    Sanger Sequencing - Human HTT knockout HeLa cell line (ab265976)
    Homozygous: 1 bp deletion in exon 21.
  • Cell Culture - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)
    Cell Culture - Human HTT (Huntingtin) knockout HeLa cell line (ab265976)

    Representative images of HTT knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.

     

     

Protocols

  • Hemocytometer protocol
  • Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

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ab265976 has not yet been referenced specifically in any publications.

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