Human CLOCK knockout HCT116 cell line
- Advanced Validation
- What is this?
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- NGS
Supplier Data
Next Generation Sequencing - Human CLOCK knockout HCT116 cell line (AB287325)
1 bp deletion and 1 bp insertion after Asp203 (edit 1); 1 bp insertion and 2 bp deletion after Lys205 (edit 2); 1 bp insertion and 1 bp insertion Lys205 (edit 3); 1 bp deletion and 2 bp deletion after Asp203 (edit 4)
- WB
Lab
Western blot - Human CLOCK knockout HCT116 cell line (AB287325)
Western blot : Anti-KAT13D / CLOCK antibody ab93804 staining at 1/2000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 105 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in CLOCK knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 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 HCT 116 whole cell lysate at 20 µg
Lane 2:
Western blot - Human CLOCK knockout HCT116 cell line (ab287325) at 20 µg
Lane 3:
HeLa whole cell lysate at 20 µg
Lane 4:
Ramos whole cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Predicted band size: 100 kDa
Observed band size: 105 kDa
false
- WB
Lab
Western blot - Human CLOCK knockout HCT116 cell line (AB287325)
Western blot : Anti-CLOCK antibody [HL1099] ab308571 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin ab7291 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 100 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in CLOCK knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes:
Western blot - Anti-CLOCK antibody [HL1099] (<a href='/en-us/products/primary-antibodies/clock-antibody-hl1099-ab308571'>ab308571</a>) at 1/1000 dilution
Lane 1:
Wild-type HCT 116 at 20 µg
Lane 2:
Western blot - Human CLOCK knockout HCT116 cell line (ab287325) at 20 µg
Lane 3:
HeLa at 20 µg
Lane 4:
Ramos at 20 µg
Secondary
All lanes:
Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Predicted band size: 95 kDa
Observed band size: 100 kDa,51 kDa
false
Complementary Products
Reactivity data
Product details
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
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
McCoY5a + 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: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
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