Pre-made Human ATG16L1 knockout THP-1 cell line (ab277834) (2024)

Overview

• Product name

  Human ATG16L1 knockout THP-1 cell line
  See all ATG16L1 lysates

• Parental Cell Line

  THP-1

• Organism

  Human

• Passage number

  <20

• Knockout validation

  Western Blot (WB)

• Tested applications

  Suitable for: WBmore details

• Biosafety level

  1

• General notes

  Recommended control:Human wild-type THP-1 cell line (ab281894). 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:RPMI + 10% FBS + 0.05 mM β-mercaptoethanol

  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-warmedculture medium, wash vial with an additional 0.8 mLculture medium(total volume 10 mL) to collect remaining cells, and centrifuge at 201x 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-warmedculture mediumand 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 2-4x10⁵cells/mL. 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.
  5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x10⁵ cells/mL.

  Subculture guidelines:

  • All seeding densities should be based on cell counts gained by established methods.
  • Cells should be seeded at 2-4x10⁵cells/mL and subcultured when they have reached 8x10⁵ cells/mL. It is not recommended to allow the cell density to exceed 1x10⁶ cells/mL.
  • 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.

  We will provide viable cells that proliferate on revival.

Properties

• Number of cells

  1 x 10⁶ cells/vial, 1 mL

• Adherent /Suspension

  Suspension

• Tissue

  Blood

• Cell type

  acute monocytic leukemia

• Disease

  Acute Monocytic Leukemia

• Gender

  Male

• 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

  • Cell Biology
  • Proteolysis / Ubiquitin
  • Proteasome / Ubiquitin
  • Ub-like Proteins

  • Neuroscience
  • Neurology process
  • Neurodegenerative disease
  • Other

  • Cancer
  • Oncoproteins/suppressors
  • Tumor suppressors
  • Other

  • Cardiovascular
  • Heart
  • Autophagy
  • APG gene products

  • Cancer
  • Signal transduction
  • Autophagy

  • Metabolism
  • Pathways and Processes
  • Metabolism processes
  • Autophagy and mitophagy

  • Metabolism
  • Pathways and Processes
  • Metabolism processes
  • Autophagy and mitophagy
  • APG gene products

  • Cancer
  • Cell Death
  • Autophagy
  • Signal Transduction

  • Cancer
  • Cell Death
  • Autophagy
  • APG gene products

  • Neuroscience
  • Processes

Target

• Function

  Plays an essential role in autophagy: interacts with ATG12-ATG5 to mediate the conjugation of phosphatidylethanolamine (PE) to LC3 (MAP1LC3A, MAP1LC3B or MAP1LC3C), to produce a membrane-bound activated form of LC3 named LC3-II. Thereby, controls the elongation of the nascent autophagosomal membrane.

• Involvement in disease

  Inflammatory bowel disease 10

• Sequence similarities

  Belongs to the WD repeat ATG16 family.
  Contains 7 WD repeats.

• Post-translational
  modifications

  Proteolytic cleavage by activated CASP3 leads to degradation and may regulate autophagy upon cellular stress and apoptotic stimuli.

• Cellular localization

  Cytoplasm. Preautophagosomal structure membrane. Recruited to omegasomes membranes by WIPI2. Omegasomes are endoplasmic reticulum connected strutures at the origin of preautophagosomal structures. Localized to preautophagosomal structure (PAS) where it is involved in the membrane targeting of ATG5. Localizes also to discrete punctae along the ciliary axoneme.

• Information by UniProt

• Form

  There are 4 isoforms produced by alternative splicing.

• Western blot - Human ATG16L1 knockout THP-1 cell line (ab277834)

  All lanes : Anti-ATG16L1 antibody [5H9A11] (ab233796) at 1/500 dilution

  Lane 1 : Wild-type THP-1 cell lysate
  Lane 2 : ATG16L1 knockout THP-1 cell lysate
  Lane 3 : Wild type HeLa cell lysate
  Lane 4 : ATG16L1 knockout HeLa cell lysate

  Lysates/proteins at 20 µg per lane.

  Performed under reducing conditions.

  Predicted band size: 68 kDa
  Observed band size: 68,70 kDa why is the actual band size different from the predicted?

  False colour image of Western blot: Anti-ATG16L1 antibody [5H9A11] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab233796 was shown to bind specifically to ATG16L1. A band was observed at 68/70 kDa in wild-type THP-1 cell lysates with no signal observed at this size in ATG16L1 knockout cell line ab277834 (knockout cell lysate ab278184). To generate this image, wild-type and ATG16L1 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

• Western blot - Human ATG16L1 knockout THP-1 cell line (ab277834)

  All lanes : Anti-ATG16L1 antibody [EPR15638] - N-terminal (ab187671) at 1/1000 dilution

  Lane 1 : Wild-type THP-1 cell lysate
  Lane 2 : ATG16L1 knockout THP-1 cell lysate
  Lane 3 : Wild type HeLa cell lysate
  Lane 4 : ATG16L1 knockout HeLa cell lysate

  Lysates/proteins at 20 µg per lane.

  Performed under reducing conditions.

  Predicted band size: 68 kDa
  Observed band size: 68,70 kDa why is the actual band size different from the predicted?

  False colour image of Western blot: Anti-ATG16L1 antibody [EPR15638] - N-terminal staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab187671 was shown to bind specifically to ATG16L1. A band was observed at 68/70 kDa in wild-type THP-1 cell lysates with no signal observed at this size in ATG16L1 knockout cell line ab277834 (knockout cell lysate ab278184). To generate this image, wild-type and ATG16L1 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

• Hemocytometer protocol
• Mammalian cell tissue culture techniques protocol

Click here to view the general protocols

• SDS download

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• Datasheet download

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ab277834 has not yet been referenced specifically in any publications.