Human PKP3 (Plakophilin 3) knockout HeLa cell line (ab265539)
製品の概要
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製品名
Human PKP3 (Plakophilin 3) knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 5 bp deletion in exon 1 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Biosafety level
2 -
特記事項
Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture 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.
4. Incubate the culture at 37°C incubator with 5% CO2. 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.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
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.
We will provide viable cells that proliferate on revival.
製品の特性
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
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 -
Mycoplasma free
Yes -
保存方法
Shipped on Dry Ice. Store in liquid nitrogen. -
バッファー
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究分野
ターゲット情報
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機能
May play a role in junctional plaques. -
組織特異性
Found in desmosomes of most simple and stratified epithelia. Not found in foreskin fibroblasts and various sarcoma-derived cell lines. Beside dendritic reticular cells of lymphatic follicles not found in non-epithelial desmosome-bearing tissues. -
配列類似性
Belongs to the beta-catenin family.
Contains 8 ARM repeats. -
細胞内局在
Nucleus. Cell junction > desmosome. Nuclear and associated with desmosomes. - Information by UniProt
関連製品
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KO cell lysates
画像
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All lanes : Anti-Plakophilin 3 antibody [EPR5560] (ab109441) at 1/10000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PKP3 CRISPR-Cas9 edited HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate
Lysates/proteins at 20 µg per lane.
Observed band size: 70, 80 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-Plakophilin 3 antibody [EPR5560] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109441 was shown to bind specifically to Plakophilin 3. A band was observed at 70 and 80 kDa in wild-type HeLa cell lysates with no signal observed at this size in PKP3 CRISPR-Cas9 edited cell line ab265539 (CRISPR-Cas9 edited cell lysate ab258120). The band observed in the CRISPR-Cas9 edited lysate lane below 70 and 80 kDa is likely to represent a truncated form of Plakophilin 3. 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 PKP3 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 3 % 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.
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Homozygous: 5 bp deletion in exon 1.
プロトコール
データシートおよび資料
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SDS download
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Datasheet download
参考文献 (0)
ab265539 は論文での使用が確認できていません。