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  1. Link

    human-clock-kat13d-knockout-hela-cell-line-ab265301.pdf

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Neuroscience Neurology process Circadian Rhythm
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Human CLOCK (KAT13D) knockout HeLa cell line (ab265301)

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Western blot - Human CLOCK (KAT13D) knockout HeLa cell line (ab265301)
  • Sanger Sequencing - Human CLOCK knockout HeLa cell line (ab265301)

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Overview

  • Product name

    Human CLOCK (KAT13D) knockout HeLa cell line
    See all KAT13D / CLOCK lysates
  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 11
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing
  • Tested applications

    Suitable for: WBmore details
  • Biosafety level

    2
  • General notes

    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
  • Mycoplasma free

    Yes
  • Storage instructions

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

    Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether
  • Research areas

    • Neuroscience
    • Neurology process
    • Circadian Rhythm
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Neuroscience
    • Neurology process
    • Metabolism
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Other factors
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Transcription Factors
    • Epigenetics and Nuclear Signaling
    • Chromatin Modifying Enzymes
    • Acetylation
    • HAT
    • Cardiovascular
    • Atherosclerosis
    • Lipoprotein metabolism
    • Cardiovascular
    • Blood
    • Blood Pressure regulation
    • Cardiovascular
    • Heart
    • Cardiac metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Lipoprotein metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
    • Metabolism
    • Types of disease
    • Heart disease

Target

  • Function

    ARNTL/2-CLOCK heterodimers activate E-box element (3'-CACGTG-5') transcription of a number of proteins of the circadian clock. Activates transcription of PER1 and PER2. This transcription is inhibited in a feedback loop by PER and CRY proteins. Has intrinsic histone acetyltransferase activity and this enzymatic function contributes to chromatin-remodeling events implicated in circadian control of gene expression (By similarity). Acetylates primarily histones H3 and H4 (By similarity). Acetylates also a non-histone substrate: ARNTL.
  • Tissue specificity

    Expressed in all tissues examined including spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Highest levels in testis and skeletal muscle. Low levels in thymus, lung and liver. Expressed in all brain regions with highest levels in cerebellum. Highly expressed in the suprachiasmatic nucleus (SCN).
  • Sequence similarities

    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • Post-translational
    modifications

    Phosphorylation is dependent on CLOCK-ARNTL heterodimer formation.
  • Cellular localization

    Cytoplasm. Nucleus. Shuffling between the cytoplasm and the nucleus is under circadian regulation and is ARNTL-dependent. Phosphorylated form located in the nucleus.
  • Target information above from: UniProt accession O15516 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 CLOCK (KAT13D) knockout HeLa cell lysate (ab258364)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab265301 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: 95 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 95 kDa.

Images

  • Western blot - Human CLOCK (KAT13D) knockout HeLa cell line (ab265301)
    Western blot - Human CLOCK (KAT13D) knockout HeLa cell line (ab265301)
    All lanes : Anti-KAT13D / CLOCK antibody (ab93804) at 1/2000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : CLOCK knockout HeLa cell lysate
    Lane 3 : A431 cell lysate
    Lane 4 : U-251 MG cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 95 kDa
    Observed band size: 100 kDa why is the actual band size different from the predicted?



    False colour image of Western blot: Anti-KAT13D / CLOCK antibody staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab93804 was shown to bind specifically to KAT13D / CLOCK. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in CLOCK knockout cell line ab265301 (knockout cell lysate ab258364). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of KAT13D / CLOCK. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CLOCK knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

  • Sanger Sequencing - Human CLOCK knockout HeLa cell line (ab265301)
    Sanger Sequencing - Human CLOCK knockout HeLa cell line (ab265301)
    Homozygous: 1 bp deletion in exon 11.

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)

Publishing research using ab265301? Please let us know so that we can cite the reference in this datasheet.

ab265301 has not yet been referenced specifically in any publications.

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