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    human-gpi-amf-knockout-hek-293t-cell-lysate-ab257458.pdf

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Human GPI (AMF) knockout HEK-293T cell lysate (ab257458)

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Western blot - Human GPI (AMF) knockout HEK293T cell lysate (ab257458)
  • Western blot - Human GPI knockout HEK293T cell lysate (ab257458)
  • Sanger Sequencing - Human GPI knockout HEK293T cell lysate (ab257458)

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Overview

  • Product name

    Human GPI (AMF) knockout HEK-293T cell lysate
    See all AMF kits
  • Product overview


    Knockout cell lysate achieved by CRISPR/Cas9.

  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 76 bp deletion in exon2.
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing, Western Blot (WB)
  • Reconstitution notes

    To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.

    *Usage of SDS sample buffer is not recommended with these lyophilized lysates.

  • Notes

    Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.

    User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

    Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
    See here for more information on knockout cell lysates.

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

    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.

  • Tested applications

    Suitable for: WBmore details

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 1 kit
    ab260993 - Human GPI knockout HEK293T cell lysate 1 x 100µg
    ab255553 - Human wild-type HEK293T cell lysate 1 x 100µg
  • Research areas

    • Neuroscience
    • Neurology process
    • Growth and Development
    • Neurotrophins
    • Immunology
    • Immune System Diseases
    • Autoimmune
    • Signal Transduction
    • Metabolism
    • Energy Metabolism
    • Cancer
    • Cancer Metabolism
    • Metabolic signaling pathway
    • Metabolism of carbohydrates
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Carbohydrate metabolism
    • Metabolism
    • Pathways and Processes
    • Metabolic signaling pathways
    • Energy transfer pathways
    • Energy Metabolism
  • 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

Target

  • Function

    Besides it's role as a glycolytic enzyme, mammalian GPI can function as a tumor-secreted cytokine and an angiogenic factor (AMF) that stimulates endothelial cell motility. GPI is also a neurotrophic factor (Neuroleukin) for spinal and sensory neurons.
  • Pathway

    Carbohydrate degradation; glycolysis; D-glyceraldehyde 3-phosphate and glycerone phosphate from D-glucose: step 2/4.
  • Involvement in disease

    Defects in GPI are the cause of hemolytic anemia non-spherocytic due to glucose phosphate isomerase deficiency (HA-GPID) [MIM:613470]. It is a form of anemia in which there is no abnormal hemoglobin or spherocytosis. It is caused by glucose phosphate isomerase deficiency. Severe GPI deficiency can be associated with hydrops fetalis, immediate neonatal death and neurological impairment.
  • Sequence similarities

    Belongs to the GPI family.
  • Post-translational
    modifications

    Phosphorylation at Ser-185 by CK2 has been shown to decrease enzymatic activity and may contribute to secretion by a non-classical secretory pathway.
    ISGylated.
  • Cellular localization

    Cytoplasm. Secreted.
  • Target information above from: UniProt accession P06744 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • AMF
    • Aurocrine motility factor
    • Autocrine motility factor
    • DKFZp686C13233
    • EC 5.3.1.9
    • G6PI_HUMAN
    • Glucose phosphate isomerase
    • Glucose-6-phosphate isomerase
    • GNPI
    • GPI
    • Gpi1
    • Hexose monophosphate isomerase
    • Hexosephosphate isomerase
    • Neuroleukin
    • NLK
    • Oxoisomerase
    • PGI
    • PHI
    • Phosphoglucose isomerase
    • Phosphohexomutase
    • Phosphohexose isomerase
    • Phosphosaccharomutase
    • SA 36
    • SA-36
    • SA36
    • Sperm antigen 36
    see all

Associated products

  • KO cell lines

    • Human GPI (AMF) knockout HEK-293T cell line (ab266834)
  • KO cell pellets

    • Human GPI (AMF) knockout HEK-293T cell pellet (ab278990)
  • Related Products

    • Anti-AMF antibody [EPR11663(B)] (ab167394)
    • Anti-AMF antibody [EPR11663(B)] - BSA and Azide free (ab231691)
    • Anti-AMF antibody [1B7D7] (ab66340)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab257458 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: 63 kDa.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 63 kDa.

Images

  • Western blot - Human GPI (AMF) knockout HEK293T cell lysate (ab257458)
    Western blot - Human GPI (AMF) knockout HEK293T cell lysate (ab257458)

    Lane 1: wild-type HEK-293T cell lysate 20 ug
    Lane 2: GPI knockout HEK-293T cell lysate 20 ug
    Lanes 1 - 2:Merged signal (red and green). Green - ab66340 observed at 63 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.
    ab66340 was shown to react with AMF in wild-type HEK-293T cells in Western blot with loss of signal observed in GPI knockout cell line ab266834 (GPI knockout cell lysate ab257458). Wild-type HEK-293T and GPI knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab66340 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Western blot - Human GPI knockout HEK293T cell lysate (ab257458)
    Western blot - Human GPI knockout HEK293T cell lysate (ab257458)

    Lane 1:Wild-type HEK293T cell lysate (20 ug)
    Lane 2:GPI knockout HEK293T cell lysate (20 ug)
    Lane 3:HepG2 cell lysate (20 ug)
    Lane 4:HeLa cell lysate (20 ug)

    ab167394 was shown to specifically react with AMF in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266834 (knockout cell lysate ab257458) was used. Wild-type and AMF knockout samples were subjected to SDS-PAGE. ab167394 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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 GPI knockout HEK293T cell lysate (ab257458)
    Sanger Sequencing - Human GPI knockout HEK293T cell lysate (ab257458)
    Homozygous: 76 bp deletion in exon2

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab257458? Please let us know so that we can cite the reference in this datasheet.

ab257458 has not yet been referenced specifically in any publications.

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