Human CLOCK (KAT13D) knockout HeLa cell lysate (ab258364)
Overview
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Product name
Human CLOCK (KAT13D) knockout HeLa cell lysate
See all KAT13D / CLOCK kits -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon11. -
Passage number
<20 -
Knockout validation
Sanger Sequencing -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.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.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab261217 - Human CLOCK knockout HeLa cell lysate 1 x 100µg ab255929 - Human wild-type HeLa cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
Target
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Function
ARNTL/2-CLOCK heterodimers activate E-box element (3'-CACGTG-5') transcription of a number of proteins of the circadian clock. Activates transcription of PER1 and PER2. This transcription is inhibited in a feedback loop by PER and CRY proteins. Has intrinsic histone acetyltransferase activity and this enzymatic function contributes to chromatin-remodeling events implicated in circadian control of gene expression (By similarity). Acetylates primarily histones H3 and H4 (By similarity). Acetylates also a non-histone substrate: ARNTL. -
Tissue specificity
Expressed in all tissues examined including spleen, thymus, prostate, testis, ovary, small intestine, colon, leukocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. Highest levels in testis and skeletal muscle. Low levels in thymus, lung and liver. Expressed in all brain regions with highest levels in cerebellum. Highly expressed in the suprachiasmatic nucleus (SCN). -
Sequence similarities
Contains 1 basic helix-loop-helix (bHLH) domain.
Contains 1 PAC (PAS-associated C-terminal) domain.
Contains 2 PAS (PER-ARNT-SIM) domains. -
Post-translational
modificationsPhosphorylation is dependent on CLOCK-ARNTL heterodimer formation. -
Cellular localization
Cytoplasm. Nucleus. Shuffling between the cytoplasm and the nucleus is under circadian regulation and is ARNTL-dependent. Phosphorylated form located in the nucleus. - Information by UniProt
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Alternative names
- bHLHe8
- Circadian locomoter output cycles kaput protein
- Circadian locomoter output cycles protein kaput
see all
Associated products
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KO cell lines
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab258364 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. |
Images
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Western blot - Human CLOCK (KAT13D) knockout HeLa cell lysate (ab258364)
Lane 1: Wild-type HeLa cell lysate 20 μg
Lane 2: CLOCK knockout HeLa cell lysate 20 μg
Lane 3: A431 cell lysate 20 μg
Lane 4: U-251 MG cell lysate 20 μg
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. -
Western blot - Human CLOCK (KAT13D) knockout HeLa cell lysate (ab258364)Lane 1: Wild-type HeLa cell lysate 20 µg
Lane 2: CLOCK CRISPR-Cas9 edited HeLa cell lysate 20 µg
Lane 3: A431 cell lysate 20 µg
Lane 4: U-251 MG cell lysate 20 µg
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 CRISPR-Cas9 edited cell line ab265301 (CRISPR-Cas9 edited cell lysate ab258364). The band observed in the CRISPR-Cas9 edited 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 CRISPR-Cas9 edited 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. -
Sanger Sequencing - Human CLOCK knockout HeLa cell lysate (ab258364)Homozygous: 1 bp deletion in exon11
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab258364 has not yet been referenced specifically in any publications.