Human ATG4A knockout HeLa cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human ATG4A knockout HeLa cell line (AB265738)
Lanes 1 - 4 : Merged signal (red and green). Green - ab108322 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab108322 was shown to react with ATG4A in wild-type HeLa cells in Western blot with loss of signal observed in ATG4A knockout cell line ab265738 (ATG4A knockout cell lysate ab257846). Wild-type HeLa and ATG4A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab108322 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:
Western blot - Anti-ATG4A antibody [EPR4122] (<a href='/en-us/products/primary-antibodies/atg4a-antibody-epr4122-ab108322'>ab108322</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
ATG4A knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human ATG4A knockout HeLa cell line (ab265738)
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
Daudi cell lysate at 20 µg
Predicted band size: 45 kDa
Observed band size: 45 kDa
false
- WB
Lab
Western blot - Human ATG4A knockout HeLa cell line (AB265738)
Lanes 1 - 4 : Merged signal (red and green). Green - ab223374 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab223374 was shown to react with ATG4A in wild-type cells in Western blot with loss of signal observed in ATG4A knockout cell line ab265738 (ATG4A knockout cell lysate ab257846). Wild-type and ATG4A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab223374 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:
Western blot - Anti-ATG4A antibody (<a href='/en-us/products/primary-antibodies/atg4a-antibody-ab223374'>ab223374</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
ATG4A knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human ATG4A knockout HeLa cell line (ab265738)
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
Daudi cell lysate at 20 µg
Predicted band size: 45 kDa
Observed band size: 45 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human ATG4A knockout HeLa cell line (AB265738)
Homozygous : 1 bp deletion in exon 4.
Complementary Products
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATG4A antibody [EPR4122] (AB108322)](https://content.abcam.com/products/images/atg4a-antibody-epr4122-ab108322--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img133664.jpg)
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
ATG4A contributes essential functions to cellular homeostasis by participating in autophagosome formation. As part of the group of Atg proteins it collaborates with other members to modulate the autophagy machinery. ATG4A's enzymatic activity provides the necessary steps for processing LC3 transforming it into a form that allows lipidation and incorporation into autophagic vesicles.
Pathways
ATG4A is integral to the autophagy pathway a catabolic process important for cell survival under stress conditions. It interacts with proteins such as ATG7 and ATG3 within this pathway playing a significant role in the early stages of autophagosome maturation. The interactions ensure proper recycling of cellular materials and its efficient function impact cellular metabolism and stress responses.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com