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    human-gba-knockout-hela-cell-line-ab265038.pdf

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Human GBA knockout HeLa cell line (ab265038)

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Sanger Sequencing - Human GBA knockout HeLa cell line (ab265038)
  • Western blot - Human GBA knockout HeLa cell line (ab265038)
  • Western blot - Human GBA knockout HeLa cell line (ab265038)
  • Sanger Sequencing - Human GBA knockout HeLa cell line (ab265038)
  • Cell Culture - Human GBA knockout HeLa cell line (ab265038)

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Overview

  • Product name

    Human GBA knockout HeLa cell line
  • Parental Cell Line

    HeLa
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 3
  • 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 HeLa cell line (ab255448). 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

    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
  • Research areas

    • Neuroscience
    • Neurology process
    • Neurodegenerative disease
    • Parkinson's disease
    • Other
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Signal Transduction
    • Metabolism
    • Lipid metabolism
    • Cancer
    • Cancer Metabolism
    • Metabolic signaling pathway
    • Metabolism of lipids and lipoproteins
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Lipid and lipoprotein metabolism
    • Lipid metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
    • Metabolism
    • Types of disease
    • Neurodegenerative disease
    • Neuroscience
    • Processes

Target

  • Involvement in disease

    Defects in GBA are the cause of Gaucher disease (GD) [MIM:230800]; also known as glucocerebrosidase deficiency. GD is the most prevalent lysosomal storage disease, characterized by accumulation of glucosylceramide in the reticulo-endothelial system. Different clinical forms are recognized depending on the presence (neuronopathic forms) or absence of central nervous system involvement, severity and age of onset.
    Defects in GBA are the cause of Gaucher disease type 1 (GD1) [MIM:230800]; also known as adult non-neuronopathic Gaucher disease. GD1 is characterized by hepatosplenomegaly with consequent anemia and thrombopenia, and bone involvement. The central nervous system is not involved.
    Defects in GBA are the cause of Gaucher disease type 2 (GD2) [MIM:230900]; also known as acute neuronopathic Gaucher disease. GD2 is the most severe form and is universally progressive and fatal. It manifests soon after birth, with death generally occurring before patients reach two years of age.
    Defects in GBA are the cause of Gaucher disease type 3 (GD3) [MIM:231000]; also known as subacute neuronopathic Gaucher disease. GD3 has central nervous manifestations.
    Defects in GBA are the cause of Gaucher disease type 3C (GD3C) [MIM:231005]; also known as pseudo-Gaucher disease or Gaucher-like disease.
    Defects in GBA are the cause of Gaucher disease perinatal lethal (GDPL) [MIM:608013]. It is a distinct form of Gaucher disease type 2, characterized by fetal onset. Hydrops fetalis, in utero fetal death and neonatal distress are prominent features. When hydrops is absent, neurologic involvement begins in the first week and leads to death within 3 months. Hepatosplenomegaly is a major sign, and is associated with ichthyosis, arthrogryposis, and facial dysmorphism.
    Note=Perinatal lethal Gaucher disease is associated with non-immune hydrops fetalis, a generalized edema of the fetus with fluid accumulation in the body cavities due to non-immune causes. Non-immune hydrops fetalis is not a diagnosis in itself but a symptom, a feature of many genetic disorders, and the end-stage of a wide variety of disorders.
    Defects in GBA contribute to susceptibility to Parkinson disease (PARK) [MIM:168600]. A complex neurodegenerative disorder characterized by bradykinesia, resting tremor, muscular rigidity and postural instability. Additional features are characteristic postural abnormalities, dysautonomia, dystonic cramps, and dementia. The pathology of Parkinson disease involves the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies (intraneuronal accumulations of aggregated proteins), in surviving neurons in various areas of the brain. The disease is progressive and usually manifests after the age of 50 years, although early-onset cases (before 50 years) are known. The majority of the cases are sporadic suggesting a multifactorial etiology based on environmental and genetic factors. However, some patients present with a positive family history for the disease. Familial forms of the disease usually begin at earlier ages and are associated with atypical clinical features.
  • Sequence similarities

    Belongs to the glycosyl hydrolase 30 family.
  • Cellular localization

    Lysosome membrane. Interaction with saposin-C promotes membrane association.
  • Target information above from: UniProt accession P04062 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 GBA knockout HeLa cell lysate (ab256929)
  • KO cell pellets

    • Human GBA knockout HeLa cell pellet (ab278951)
  • Related Products

    • Anti-GBA antibody [EPR5142] (ab125065)
    • Anti-GBA antibody [EPR5143(3)] (ab128879)
    • Anti-GBA antibody [EPR5143(3)] - BSA and Azide free (ab215260)
    • Anti-GBA antibody [EPR5142] - BSA and Azide free (ab215261)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab265038 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: 60 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 60 kDa.

Images

  • Sanger Sequencing - Human GBA knockout HeLa cell line (ab265038)
    Sanger Sequencing - Human GBA knockout HeLa cell line (ab265038)
    Sequencing chromatogram displaying sequence edit in exon 3
  • Western blot - Human GBA knockout HeLa cell line (ab265038)
    Western blot - Human GBA knockout HeLa cell line (ab265038)
    All lanes : Anti-GBA antibody [EPR5143(3)] (ab128879) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : GBA knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 60 kDa
    Observed band size: 60 kDa



    Lanes 1- 2: Merged signal (red and green). Green - ab128879 observed at 60 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab128879 was shown to react with GBA in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265038 (knockout cell lysate ab256929) was used. Wild-type HeLa and GBA 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. ab128879 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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 GBA knockout HeLa cell line (ab265038)
    Western blot - Human GBA knockout HeLa cell line (ab265038)
    All lanes : Anti-GBA antibody [EPR5142] (ab125065) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : GBA knockout HeLa cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 60 kDa
    Observed band size: 70 kDa why is the actual band size different from the predicted?



    Lanes 1- 2: Merged signal (red and green). Green - ab125065 observed at 70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

     ab125065 was shown to react with GBA in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265038 (knockout cell lysate ab256929) was used. Wild-type HeLa and GBA 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. ab125065 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 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 GBA knockout HeLa cell line (ab265038)
    Sanger Sequencing - Human GBA knockout HeLa cell line (ab265038)
    Homozygous: 1 bp insertion in exon 3.
  • Cell Culture - Human GBA knockout HeLa cell line (ab265038)
    Cell Culture - Human GBA knockout HeLa cell line (ab265038)

    Representative images of GBA 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)

Publishing research using ab265038? Please let us know so that we can cite the reference in this datasheet.

ab265038 has not yet been referenced specifically in any publications.

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