Human ANXA6 (Annexin-6) knockout HeLa cell line (ab265677)

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Overview

  • Product name

    Human ANXA6 (Annexin-6) knockout HeLa cell line

  • Parental Cell Line

    HeLa

  • Organism

    Human

  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 7 and 5 bp deletion in exon 7

  • 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 (ab255928)

    Please note a wild type cell line is not automatically included with a KO 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, supplemented with 10% (v/v) DMSO

    Handling procedure: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C will result in loss of viability.
    1. Thaw the vial in 37°C water bath approximately 1-2 minutes.
    2. Transfer the cell suspension to a 15 mL conical tube with pre-warmed 5 mL complete medium DMEM+10% FBS, spin 125×g for approximately 5 minutes at room temperature.
    3. Resuspend the cell pellet with 1 mL pre-warmed complete medium DMEM+10% FBS and dispense into a 25 cm2 culture flask containing 10 mL pre-warmed complete complete medium DMEM+10% FBS.
    4. Incubate the culture at 37°C incubator with 5% CO2.
    5. A subcultivation ratio of 1:4-1:6 is recommended. Cells should be passaged when cells grow splitting at 80-90% confluence.

    Click here to view the Mammalian cell tissue culture protocol

    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

Target

  • Function

    May associate with CD21. May regulate the release of Ca(2+) from intracellular stores.

  • Sequence similarities

    Belongs to the annexin family.
    Contains 8 annexin repeats.

  • Domain

    A pair of annexin repeats may form one binding site for calcium and phospholipid.

  • Post-translational
    modifications

    Phosphorylated in response to growth factor stimulation.

  • Cellular localization

    Cytoplasm. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt

Applications

Our Abpromise guarantee covers the use of ab265677 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: 76 kDa.

Images

  • Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
    Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
    All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19517] (ab201023) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : ANXA6 knockout HeLa cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : Raji cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

    Predicted band size: 76 kDa
    Observed band size: 75 kDa
    why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab201023 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

     ab201023 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with 6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used.  Wild-type and 6/ANXA6 knockout samples were subjected to SDS-PAGE.  ab201023 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.

  • Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
    Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
    All lanes : Anti-Annexin-6/ANXA6 antibody [EPR19536] (ab201024) at 1/1000 dilution

    Lane 1 : Wild-type HeLa cell lysate
    Lane 2 : ANXA6 knockout HeLa cell lysate
    Lane 3 : Jurkat cell lysate
    Lane 4 : Raji cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

    Predicted band size: 76 kDa
    Observed band size: 75 kDa why is the actual band size different from the predicted?



    Lanes 1-4: Merged signal (red and green). Green - ab201024 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa. 

     ab201024 Anti-Annexin-6/ANXA6 antibody [EPR19536] was shown to specifically react with Annexin-6/ANXA6 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265677 (knockout cell lysate ab257351) was used.  Wild-type and Annexin-6/ANXA6 knockout samples were subjected to SDS-PAGE.  ab201024 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 ANXA6 knockout HeLa cell line (ab265677)
    Sanger Sequencing - Human ANXA6 knockout HeLa cell line (ab265677)
    Allel-1: 5 bp deletion in exon 7.
  • Sanger Sequencing - Human ANXA6 knockout HeLa cell line (ab265677)
    Sanger Sequencing - Human ANXA6 knockout HeLa cell line (ab265677)
    Allel-2: 1 bp insertion in exon 7.

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

References

Publishing research using ab265677? Please let us know so that we can cite the reference in this datasheet.

ab265677 has not yet been referenced specifically in any publications.

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