Human CLOCK (KAT13D) knockout HeLa cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human CLOCK (KAT13D) knockout HeLa cell line (AB265301)
False colour image of Western blot : Anti-KAT13D / CLOCK antibody staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab93804 was shown to bind specifically to KAT13D / CLOCK. A band was observed at 100 kDa in wild-type HeLa cell lysates with no signal observed at this size in CLOCK knockout cell line ab265301 (knockout cell lysate ab258364). The band observed in the knockout lysate lane below 100 kDa is likely to represent a truncated form of KAT13D / CLOCK. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CLOCK 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-KAT13D / CLOCK antibody (<a href='/en-us/products/primary-antibodies/kat13d-clock-antibody-ab93804'>ab93804</a>) at 1/2000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
CLOCK knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human CLOCK (KAT13D) knockout HeLa cell line (ab265301)
Lane 3:
A431 cell lysate at 20 µg
Lane 4:
U-251 MG cell lysate at 20 µg
Predicted band size: 95 kDa
Observed band size: 100 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human CLOCK (KAT13D) knockout HeLa cell line (AB265301)
Homozygous : 1 bp deletion in exon 11.
Complementary Products
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
The CLOCK protein acts as an important component of the circadian rhythm machinery. It forms a heterodimer complex with BMAL1 which activates transcription of other core clock genes. This process drives the rhythmic expression of various genes essential for physiological and behavioral rhythms. Through this function CLOCK influences the timing of many body systems such as sleep-wake cycles feeding and metabolism. By doing so it sets a steady rhythm to coordinate bodily processes with environmental light-dark cycles ensuring optimal biological activity during appropriate times of the day.
Pathways
The CLOCK protein plays an important role in the circadian signaling pathway where its function involves intricate feedback loops. It controls the oscillation of gene expression alongside other clock proteins like PER and CRY. This feedback mechanism is part of the circadian rhythm regulation pathway which directly influences processes such as hormone regulation and cell cycle progression. CLOCK's relationship with BMAL1 PER and CRY in these pathways highlights its indispensable role in maintaining the synchronization of endogenous biological rhythms with external time cues.
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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