Human PDK4 knockout HeLa cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Lab
Western blot - Human PDK4 knockout HeLa cell line (AB261805)
Lanes 1-3 : Merged signal (red and green). Green - ab110336 observed at 48 kDa. Red - loading control, ab129002 observed at 124 kDa.
ab110336 Anti-PDK4 antibody [1C2BG5] was shown to specifically react with PDK4 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261805 (knockout cell lysate ab257217) was used. Wild-type and PDK4 knockout samples were subjected to SDS-PAGE. ab110336 and Anti-Vinculin antibody [EPR8185] - Loading Control (ab129002) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 1000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-PDK4 antibody [1C2BG5] (<a href='/en-us/products/primary-antibodies/pdk4-antibody-1c2bg5-ab110336'>ab110336</a>) at 1/500 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
PDK4 knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human PDK4 knockout HeLa cell line (ab261805)
Lane 3:
Human heart tissue lysate at 20 µg
Predicted band size: 46 kDa
Observed band size: 48 kDa
false
- WB
Lab
Western blot - Human PDK4 knockout HeLa cell line (AB261805)
Western blot : Anti-PDK4 antibody [1C2BG5] (ab110336) staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab110336 was shown to bind specifically to PDK4. A band was observed at 45 kDa in wild-type HeLa cell lysates with no signal observed at this size in PDK4 knockout cell line. To generate this image, wild-type and PDK4 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
All lanes:
Western blot - Anti-PDK4 antibody [1C2BG5] (<a href='/en-us/products/primary-antibodies/pdk4-antibody-1c2bg5-ab110336'>ab110336</a>) at 1 µg/mL
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
PDK4 knockout HeLa cell lysate at 20 µg
Lane 3:
HepG2 cell lysate at 20 µg
Lane 4:
THP-1 cell lysate at 20 µg
Lane 5:
Jurkat cell lysate at 20 µg
Lane 6:
A431 cell lysate at 20 µg
Lane 7:
Human Heart cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution
Observed band size: 45 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human PDK4 knockout HeLa cell line (AB261805)
Allele-2 : 1 bp insertion in exon 1.
- Sanger seq
Unknown
Sanger Sequencing - Human PDK4 knockout HeLa cell line (AB261805)
Allele-1 : 1 bp deletion in exon 1.
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Primary Antibodies
AB179483
Anti-HIF-1 alpha antibody [EPR16897]
RabMAb|Advanced Validation|Recombinant|KO Validated
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- 4
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Cell Lines & Lysates
AB255434
Human SOD2 (MnSOD) knockout HEK-293T cell line
Advanced Validation
- 0
- 0
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, 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.
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
The enzyme functions to inhibit the pyruvate dehydrogenase complex by adding phosphate groups which reduces the conversion of pyruvate to acetyl-CoA and shifts energy production from carbohydrates to fats especially during fasting or prolonged exercise. PDK4 operates as a homodimer and is often part of a larger regulatory system balancing glucose and fatty acid oxidation in response to nutrient availability.
Pathways
PDK4 is integral to the regulation of both glucose metabolism and fatty acid oxidation. The enzyme interacts closely with the insulin signaling pathway where it modulates glucose uptake and utilization based on hormonal signals. Additionally PDK4 is a part of the hypoxia-inducible factor-1 (HIF-1) pathway where it plays a role in cellular response to low oxygen levels interacting with related proteins such as pyruvate dehydrogenase (PDH).
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