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  1. Link

    human-myc-c-myc-knockout-hek-293t-cell-line-ab256500.pdf

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Epigenetics and Nuclear Signaling Transcription Domain Families HLH / Leucine Zipper HLH / Leucine Zipper
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Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)

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  • SDS
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Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
  • Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
  • Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
  • Western blot - Human MYC knockout HEK293T cell line (ab256500)
  • Immunocytochemistry - Human MYC (c-Myc) knockout HEK293T cell line (ab256500)
  • Sanger Sequencing - Human MYC knockout HEK293T cell line (ab256500)

You may also be interested in

Protein
Recombinant human c-Myc protein (Active) (ab169901)
Cells
Human wild-type HEK-293T cell line (ab255449)
Primary
Product image
Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

View more associated products

Overview

  • Product name

    Human MYC (c-Myc) knockout HEK-293T cell line
  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous (4N): 1bp T insertion (2N); 8 bp deletion and C to T insertion (1N); 4 bp deletion in exon 2 (1N)
  • Passage number

    <20
  • Knockout validation

    Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: ICC, WBmore details
  • Biosafety level

    2
  • General notes

    Recommended control: Human wild-type HEK293T cell line (ab255449). 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and 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

    Kidney
  • Cell type

    epithelial
  • STR Analysis

    Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12
  • 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

    • Epigenetics and Nuclear Signaling
    • Transcription
    • Domain Families
    • HLH / Leucine Zipper
    • HLH / Leucine Zipper
    • Stem Cells
    • Signaling Pathways
    • TGF beta
    • Nuclear
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Transcription Factors
    • Cancer
    • Cell cycle
    • Cell differentiation
    • Cancer
    • Oncoproteins/suppressors
    • Oncoproteins
    • Transcription factors
    • Cancer
    • Tumor biomarkers
    • Oncoproteins

Target

  • Function

    Participates in the regulation of gene transcription. Binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3'. Seems to activate the transcription of growth-related genes.
  • Involvement in disease

    Note=Overexpression of MYC is implicated in the etiology of a variety of hematopoietic tumors.
    Note=A chromosomal aberration involving MYC may be a cause of a form of B-cell chronic lymphocytic leukemia. Translocation t(8;12)(q24;q22) with BTG1.
    Defects in MYC are a cause of Burkitt lymphoma (BL) [MIM:113970]. A form of undifferentiated malignant lymphoma commonly manifested as a large osteolytic lesion in the jaw or as an abdominal mass. Note=Chromosomal aberrations involving MYC are usually found in Burkitt lymphoma. Translocations t(8;14), t(8;22) or t(2;8) which juxtapose MYC to one of the heavy or light chain immunoglobulin gene loci.
  • Sequence similarities

    Contains 1 basic helix-loop-helix (bHLH) domain.
  • Post-translational
    modifications

    Phosphorylated by PRKDC. Phosphorylation at Thr-58 and Ser-62 by GSK3 is required for ubiquitination and degradation by the proteasome.
    Ubiquitinated by the SCF(FBXW7) complex when phosphorylated at Thr-58 and Ser-62, leading to its degradation by the proteasome. In the nucleoplasm, ubiquitination is counteracted by USP28, which interacts with isoform 1 of FBXW7 (FBW7alpha), leading to its deubiquitination and preventing degradation. In the nucleolus, however, ubiquitination is not counteracted by USP28, due to the lack of interaction between isoform 4 of FBXW7 (FBW7gamma) and USP28, explaining the selective MYC degradation in the nucleolus. Also polyubiquitinated by the DCX(TRUSS) complex.
  • Cellular localization

    Nucleus > nucleoplasm. Nucleus > nucleolus.
  • Target information above from: UniProt accession P01106 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Form

    c-Myc is also expressed in the cytoplasm.

Associated products

  • KO cell lysates

    • Human MYC (c-Myc) knockout HEK-293T cell lysate (ab263850)
  • KO cell pellets

    • Human MYC (c-Myc) knockout HEK-293T cell pellet (ab278810)
  • Related Products

    • Anti-c-Myc antibody [Y69] - ChIP Grade - BSA and Azide free (ab168727)
    • Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab256500 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
Use at an assay dependent concentration.
WB (1)
Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.
Notes
ICC
Use at an assay dependent concentration.
WB
Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.

Images

  • Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    All lanes : Anti-Myc tag antibody [Myc.A7] (ab18185) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : MYC knockout HEK-293T cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 48 kDa



    False colour image of Western blot: Anti-Myc tag antibody [Myc.A7] staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab18185 was shown to bind specifically to Myc tag. A band was observed at 57 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in MYC knockout cell line ab256500 (knockout cell lysate ab263850). The band observed in the knockout lysate lane below 57 kDa is likely to represent a truncated form of Myc tag. 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 MYC knockout HEK-293T cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

  • Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    All lanes : Anti-Myc tag antibody [9E10] (ab32) at 1/200 dilution

    Lane 2 : MYC knockout HEK-293T cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 48 kDa



    False colour image of Western blot: Anti-Myc tag antibody [9E10] staining at 1/200 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32 was shown to bind specifically to Myc tag. A band was observed at 57 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in MYC knockout cell line ab256500 (knockout cell lysate ab263850). The band observed in the knockout lysate lane below 57 kDa is likely to represent a truncated form of Myc tag. 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 MYC knockout HEK-293T cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

  • Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    Western blot - Human MYC (c-Myc) knockout HEK-293T cell line (ab256500)
    All lanes : Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : MYC knockout HEK-293T cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : SH-SY5Y cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 48 kDa



    False colour image of Western blot: Anti-c-Myc antibody [Y69] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32072 was shown to bind specifically to c-Myc. A band was observed at 45/57 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in MYC knockout cell line ab256500 (knockout cell lysate ab263850). The band observed in the knockout lysate lane below 45/57 kDa is likely to represent a truncated form of c-Myc. 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 MYC knockout HEK-293T 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.

  • Western blot - Human MYC knockout HEK293T cell line (ab256500)
    Western blot - Human MYC knockout HEK293T cell line (ab256500)
    All lanes : Anti-c-Myc antibody [Y69] - ChIP Grade (ab32072) at 1/1000 dilution

    Lane 1 : Jurkat cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : Wild-type HEK-293T cell lysate
    Lane 4 : MYC knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution

    Predicted band size: 48 kDa
    Additional bands at: 37 kDa (possible Loading Control)



    Lanes 1 - 4: Merged signal (red and green). Green - ab32072 observed at 57 kDa. Red - loading control, ab8245 observed at 37 kDa.

     ab32072 was shown to react with MYC in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab256500 (knockout cell lysate ab263850) was used. Wild-type and MYC knockout samples were subjected to SDS-PAGE. ab32072 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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.

     

  • Immunocytochemistry - Human MYC (c-Myc) knockout HEK293T cell line (ab256500)
    Immunocytochemistry - Human MYC (c-Myc) knockout HEK293T cell line (ab256500)
    ab32072 staining MYC in wild-type HEK293 cells (top panel) and MYC knockout HEK293 cells (ab256500) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab32072 at 5μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
    Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
  • Sanger Sequencing - Human MYC knockout HEK293T cell line (ab256500)
    Sanger Sequencing - Human MYC knockout HEK293T cell line (ab256500)
    Homozygous: 1 bp insertion in exon 2

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 ab256500? Please let us know so that we can cite the reference in this datasheet.

ab256500 has not yet been referenced specifically in any publications.

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Western blot to confirm MYC KO

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Abreviews
Abreviews
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Application
Western blot
A Western blot was performed on MYC WT and MYC KO HEK293T cells to confirm knockout of MYC protein. The primary antibody used was ab32072. Lane 1: HEK293T cells (ab255449). Lane 2: MYC KO HEK293T cells (ab256500).

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Submitted Jul 14 2021

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