Human APPL1 (APPL) knockout HeLa cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human APPL1 (APPL) knockout HeLa cell line (AB265187)
Lanes 1- 4 : Merged signal (red and green). Green - ab180140 observed at 90 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab180140 was shown to react with Anti-APPL in wild-type HeLa cells in western blot. The band observed in CRISPR/Cas9 edited cell line ab265187 (CRISPR/Cas9 edited cell lysate ab257836) lane below 90kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and APPL1 CRISPR/Cas9 edited HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab180140 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at a 1 in 10000 dilution and a 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.
All lanes:
Western blot - Anti-APPL antibody [EPR13569] (<a href='/en-us/products/primary-antibodies/appl-antibody-epr13569-ab180140'>ab180140</a>) at 1/10000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
APPL CRISPR/Cas9 edited HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human APPL1 (APPL) knockout HeLa cell line (ab265187)
Lane 3:
MCF7 cell lysate at 20 µg
Lane 4:
HEK-293T cell lysate at 20 µg
Predicted band size: 80 kDa
Observed band size: 90 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human APPL1 (APPL) knockout HeLa cell line (AB265187)
Homozygous : 2 bp deletion in exon 2.
Complementary Products
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-APPL antibody [EPR13569] (AB180140)](https://content.abcam.com/products/images/appl-antibody-epr13569-ab180140--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img64945.jpg)
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
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
APPL1 influences cellular processes such as endocytosis and cell proliferation. APPL1 interacts with rab5 forming a component of endosomal signaling complexes. This interaction drives the early endocytic process influencing cell surface receptor trafficking. APPL1 also modulates insulin signaling by binding to proteins like the insulin receptor substrate affecting glucose metabolism and cellular energy balance.
Pathways
APPL1 acts in the AKT signaling pathway and the adiponectin signaling pathway. In the AKT pathway APPL1 operates with proteins like AKT itself and GSK3β contributing to cell survival and growth. It assists the adiponectin pathway by facilitating interactions between adiponectin receptors and downstream effectors impacting lipid metabolism and insulin sensitivity. APPL1's involvement in these pathways highlights its multifaceted regulatory roles in cellular metabolism and response to stimuli.
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'.
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