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    products/cell-lines/human-uchl1-pgp95-knockout-hek-293t-cell-line-ab255443.pdf

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Human UCHL1 (PGP9.5) knockout HEK-293T cell line (ab255443)

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Western blot - Human UCHL1 (PGP9.5) knockout HEK-293T cell line (ab255443)
  • Sanger Sequencing - Human UCHL1 knockout HEK293T cell line (ab255443)

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Primary
Product image
Anti-PGP9.5 antibody [EPR4118] - Neuronal Marker (ab108986)
Cells
Human wild-type HEK-293T cell line (ab255449)
Knockout
Product image
Human ADRBK1 (GRK2) knockout HEK-293T cell line (ab266352)

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Overview

  • Product name

    Human UCHL1 (PGP9.5) knockout HEK-293T cell line
  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 45 bp deletion in exon 1
  • 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 (ab255593). 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

    Kidney
  • Cell type

    epithelial
  • STR Analysis

    Amelogenin X D5S818: 8, 9 D13S317: 11, 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 15, 20 TH01: 7, 9.3 TPOX: 11, 12 CSF1PO: 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

    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Parkinson's disease
    • Parkin / PARK
    • Neuroscience
    • Cell Type Marker
    • Neuron marker
    • Soma marker
    • Cell Biology
    • Proteolysis / Ubiquitin
    • Proteasome / Ubiquitin
    • Deubiquitination
    • Tags & Cell Markers
    • Cell Type Markers
    • Neuroscience Markers
    • Neuronal
    • Neuroscience
    • Diseases

Target

  • Function

    Ubiquitin-protein hydrolase involved both in the processing of ubiquitin precursors and of ubiquitinated proteins. This enzyme is a thiol protease that recognizes and hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin. Also binds to free monoubiquitin and may prevent its degradation in lysosomes. The homodimer may have ATP-independent ubiquitin ligase activity.
  • Tissue specificity

    Found in neuronal cell bodies and processes throughout the neocortex (at protein level). Expressed in neurons and cells of the diffuse neuroendocrine system and their tumors. Weakly expressed in ovary. Down-regulated in brains from Parkinson disease and Alzheimer disease patients.
  • Involvement in disease

    Parkinson disease 5
    Neurodegeneration with optic atrophy, childhood-onset
  • Sequence similarities

    Belongs to the peptidase C12 family.
  • Post-translational
    modifications

    O-glycosylated.
  • Cellular localization

    Cytoplasm. Endoplasmic reticulum membrane. About 30% of total UCHL1 is associated with membranes in brain.
  • Target information above from: UniProt accession P09936 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 UCHL1 (PGP9.5) knockout HEK-293T cell lysate (ab263773)
  • Related Products

    • Anti-PGP9.5 antibody [EPR4118] - Neuronal Marker (ab108986)
    • Anti-PGP9.5 antibody [EPR4118] - BSA and Azide free (ab220823)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab255443 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: 24 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 24 kDa.

Images

  • Western blot - Human UCHL1 (PGP9.5) knockout HEK-293T cell line (ab255443)
    Western blot - Human UCHL1 (PGP9.5) knockout HEK-293T cell line (ab255443)
    All lanes : Anti-PGP9.5 antibody [EPR4118] - Neuronal Marker (ab108986) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 cell lysate
    Lane 2 : UCHL1 knockout HAP1 cell lysate
    Lane 3 : SH-SY5Y cell lysate
    Lane 4 : Wild-type HEK-293T cell lysate
    Lane 5 : UCHL1 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

    Performed under reducing conditions.

    Predicted band size: 24 kDa
    Additional bands at: 37 kDa (possible Loading Control)



    Lanes 1 - 5: Merged signal (red and green). Green - ab108986 observed at 25 kDa. Red - loading control, ab8245 observed at 37 kDa.

    ab108986 was shown to react with PGP9.5 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab255443 (knockout cell lysate ab263773) was used. Wild-type and PGP9.5 knockout samples were subjected to SDS-PAGE. ab108986 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.

  • Sanger Sequencing - Human UCHL1 knockout HEK293T cell line (ab255443)
    Sanger Sequencing - Human UCHL1 knockout HEK293T cell line (ab255443)
    Homozygous: 45 bp deletion in exon 1.

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 ab255443? Please let us know so that we can cite the reference in this datasheet.

ab255443 has not yet been referenced specifically in any publications.

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