Human ATG4B knockout HeLa cell line (ab265814)
Overview
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Product name
Human ATG4B knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 4 and 1 bp insertion in exon 4 -
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
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 -
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
Target
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Function
Cysteine protease required for autophagy, which cleaves the C-terminal part of either MAP1LC3, GABARAPL2 or GABARAP, allowing the liberation of form I. A subpopulation of form I is subsequently converted to a smaller form (form II). Form II, with a revealed C-terminal glycine, is considered to be the phosphatidylethanolamine (PE)-conjugated form, and has the capacity for the binding to autophagosomes. -
Tissue specificity
Mainly expressed in the skeletal muscle, followed by brain, heart, liver and pancreas. -
Sequence similarities
Belongs to the peptidase C54 family. -
Cellular localization
Cytoplasm. - Information by UniProt
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab265814 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 |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 44 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 44 kDa. |
Images
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All lanes : Anti-ATG4B antibody [EPR16572] (ab199537) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATG4B knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?Lanes 1 - 2:Merged signal (red and green). Green - ab199537 observed at 47 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab199537 was shown to react with ATG4B in wild-type HeLa cells in Western blot with loss of signal observed in ATG4B knockout cell line ab260973 (ATG4B knockout cell lysate ab257364). Wild-type and ATG4B knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab199537 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 h at room temperature before imaging. -
All lanes : Anti-ATG4B antibody [EPR6436(2)] (ab154843) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : ATG4B knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 44 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?Lanes 1 - 2:Merged signal (red and green). Green - ab154843 observed at 47 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab154843 was shown to react with ATG4B in wild-type HeLa cells in Western blot with loss of signal observed in ATG4B knockout cell line ab260973 (ATG4B knockout cell lysate ab257364). Wild-type and ATG4B knockout HEK293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab154843 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 h at room temperature before imaging. -
Allele-1: 1 bp deletion in exon 4.
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Allele-2: 1 bp insertion in exon 4.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab265814 has not yet been referenced specifically in any publications.