Human HADHA knockout HEK-293T cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human HADHA knockout HEK-293T cell line (AB266274)
Lanes 1-4 : Merged signal (red and green). Green - ab203114 observed at 82 kDa. Red - loading control ab8245 observed at 37 kDa.
ab203114 Anti-HADHA antibody [EPR17940] was shown to specifically react with HADHA in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266274 (knockout cell lysate ab257464) was used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab203114 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 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.
All lanes:
Western blot - Anti-HADHA antibody [EPR17940] (<a href='/en-us/products/primary-antibodies/hadha-antibody-epr17940-ab203114'>ab203114</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
HADHA knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human HADHA knockout HEK-293T cell line (ab266274)
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
SH-SY5Y cell lysate at 20 µg
Predicted band size: 83 kDa
Observed band size: 82 kDa
false
- WB
Lab
Western blot - Human HADHA knockout HEK-293T cell line (AB266274)
Lanes 1-4 : Merged signal (red and green). Green - ab200652 observed at 82 kDa. Red - loading control ab8245 observed at 37 kDa.
ab200652 Anti-HADHA antibody [EPR17939] was shown to specifically react with HADHA in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266274 (knockout cell lysate ab257464) was used. Wild-type and HADHA knockout samples were subjected to SDS-PAGE. ab200652 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° at 1 in 1000 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.
All lanes:
Western blot - Anti-HADHA antibody [EPR17939] (<a href='/en-us/products/primary-antibodies/hadha-antibody-epr17939-ab200652'>ab200652</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
HADHA knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human HADHA knockout HEK-293T cell line (ab266274)
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
SH-SY5Y cell lysate at 20 µg
Predicted band size: 83 kDa
Observed band size: 82 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human HADHA knockout HEK-293T cell line (AB266274)
Allele-1 : 8 bp deletion in exon 1
- Sanger seq
Unknown
Sanger Sequencing - Human HADHA knockout HEK-293T cell line (AB266274)
Allele-2 : 1 bp deletion in exon 1.
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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
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
HADHA is a part of the mitochondrial trifunctional protein complex which consists of four alpha and four beta subunits. It facilitates the hydration of enoyl-CoA to 3-hydroxyacyl-CoA and the subsequent dehydrogenation to 3-ketoacyl-CoA. This enzyme works closely with its partner the HADHB protein to carry out these reactions efficiently. These functions are important for energy production as they are steps in the breakdown of fatty acids necessary for ATP generation.
Pathways
HADHA participates in the mitochondrial beta-oxidation pathway an essential pathway for energy production from fats. Alongside HADHB it catalyzes key reactions that allow the progressive shortening of fatty acid chains which further feeds into the citric acid cycle. This pathway links HADHA not only to HADHB but also to other enzymes involved in lipid metabolism and energy homeostasis including medium-chain specific acyl-CoA dehydrogenase (MCAD) reflecting its role in comprehensive metabolic networks.
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