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Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)

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Sanger Sequencing - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
  • Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
  • Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
  • Sanger Sequencing - Human SQSTM1 knockout HEK293T cell line (ab255343)

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Recombinant Human SQSTM1 / p62 protein (ab95320)

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Overview

  • Product name

    Human SQSTM1 (p62) knockout HEK-293T cell line
    See all SQSTM1 / p62 lysates
  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing, Western Blot (WB)
  • Tested applications

    Suitable for: 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

    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFkB Pathway
    • Signal Transduction
    • Protein Trafficking
    • Vesicle Transport
    • Regulation
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Polymerase associated factors
    • Pol II Transcription
    • Other
    • Cardiovascular
    • Heart
    • Autophagy
    • Autophagosome
    • Metabolism
    • Pathways and Processes
    • Metabolism processes
    • Autophagy and mitophagy
    • Autophagosome
    • Cancer
    • Cell Death
    • Autophagy
    • Autophagosome
    • Neuroscience
    • Processes

Target

  • Function

    Adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. May play a role in titin/TTN downstream signaling in muscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates the interaction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels.
  • Tissue specificity

    Ubiquitously expressed.
  • Involvement in disease

    Defects in SQSTM1 are a cause of Paget disease of bone (PDB) [MIM:602080]. PDB is a metabolic bone disease affecting the axial skeleton and characterized by focal areas of increased and disorganized bone turn-over due to activated osteoclasts. Manifestations of the disease include bone pain, deformity, pathological fractures, deafness, neurological complications and increased risk of osteosarcoma. PDB is a chronic disease affecting 2 to 3% of the population above the age of 40 years.
  • Sequence similarities

    Contains 1 OPR domain.
    Contains 1 UBA domain.
    Contains 1 ZZ-type zinc finger.
  • Domain

    The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinated substrates. Mediates the interaction with TRIM55.
    The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5 and NBR1.
    The ZZ-type zinc finger mediates the interaction with RIPK1.
  • Post-translational
    modifications

    Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN.
  • Cellular localization

    Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes to the sarcomeric band (By similarity). Localizes to late endosomes. May also localize to the nucleus. Accumulates in neurofibrillary tangles and in Lewy bodies of neurons from individuals with Alzheimer and Parkinson disease respectively. Enriched in Rosenthal fibers of pilocytic astrocytoma. In liver cells, accumulates in Mallory bodies associated with alcoholic hepatitis, Wilson disease, indian childhood cirrhosis and in hyaline bodies associated with hepatocellular carcinoma.
  • Target information above from: UniProt accession Q13501 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 SQSTM1 (p62) knockout HEK-293T cell lysate (ab263770)
  • Related Products

    • Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
    • Anti-SQSTM1 / p62 antibody [EPR4844] - BSA and Azide free (ab219581)
    • Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab255343 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: 47 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 47 kDa.

Images

  • Sanger Sequencing - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
    Sanger Sequencing - Human SQSTM1 (p62) knockout HEK-293T cell line (ab255343)
    Sequencing chromatogram displaying sequence edit in exon 4
  • Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
    Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
    All lanes : Anti-SQSTM1 / p62 antibody [2C11] - BSA and Azide free (ab56416) at 1/1000 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : SQSTM1 knockout HEK293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 62 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab56416 observed at 62 kDa. Red - Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) observed at 37 kDa.

     ab56416 was shown to react with SQSTM1 in wild-type HEK293T cells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate ab263770) was used. Wild-type HEK293T and SQSTM1 knockout HEK293T 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. ab56416 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  • Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
    Western blot - Human SQSTM1 (p62) knockout HEK293T cell line (ab255343)
    All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012) at 1/10000 dilution

    Lane 1 : Wild-type HEK293T cell lysate
    Lane 2 : SQSTM1 knockout HEK293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 64 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab109012 observed at 64 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab109012 was shown to react with SQSTM1 in wild-type HEK293T cells in western blot. Loss of signal was observed when knockout cell line ab255343 (knockout cell lysate ab263770) was used. Wild-type HEK293T and SQSTM1 knockout HEK293T 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. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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 SQSTM1 knockout HEK293T cell line (ab255343)
    Sanger Sequencing - Human SQSTM1 knockout HEK293T cell line (ab255343)
    Homozygous: 1 bp insertion in exon 4

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 ab255343? Please let us know so that we can cite the reference in this datasheet.

ab255343 has not yet been referenced specifically in any publications.

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