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

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Western blot - Human ANXA6 (Annexin-6) knockout HeLa cell line (ab265677)
  • Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
  • Western blot - Human ANXA6 knockout HeLa cell line (ab265677)
  • Sanger Sequencing - Human ANXA6 knockout HeLa cell line (ab265677)
  • Sanger Sequencing - Human ANXA6 knockout HeLa cell line (ab265677)

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 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

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

The Abpromise guarantee

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.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 76 kDa.

Images

  • Western blot - Human ANXA6 (Annexin-6) knockout HeLa cell line (ab265677)
    Western blot - Human ANXA6 (Annexin-6) knockout HeLa cell line (ab265677)
    All lanes : Anti-Annexin-6/ANXA6 antibody [EPR17308] - N-terminal (ab199422) 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.

    Performed under reducing conditions.

    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 - ab199422 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

    ab199422 was shown to react with Annexin-6/ANXA6 in wild-type HeLa cells in western blot with loss of signal observed in ANXA6 knockout cell line ab265677 (ANXA6 knockout cell lysate ab257351). Wild-type and ANXA6 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab199422 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (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 [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)

    Allele-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)

    Allele-2: 1 bp insertion in exon 7.

     

References (0)

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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