Human SQSTM1 (p62) knockout HCT116 cell line (ab266871)
製品の概要
-
製品名
Human SQSTM1 (p62) knockout HCT116 cell line
SQSTM1 / p62 ライゼート 製品一覧 -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 49 bp deletion in exon 4 -
Passage number
<20 -
Knockout validation
Sanger Sequencing -
アプリケーション
適用あり: WBmore details -
Biosafety level
1 -
特記事項
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
Recommended control: Human wild-type HCT116 cell line (ab255451). 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: McCoY5a + 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
-
Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Colon -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
STR Analysis
Amelogenin X D5S818: 10, 11 D13S317: 10, 12 D7S820: 11, 12 D16S539: 11, 13 vWA: 17, 22 TH01: 8,9 TPOX: 8, 9 CSF1PO: 7, 10 -
Antibiotic resistance
Puromycin 1.00µg/ml -
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
-
機能
Adapter protein which binds ubiquitin and may regulate the activation of NFKB1 by TNF-alpha, nerve growth factor (NGF) and interleukin-1. May play a role in titin/TTN downstream signaling in muscle cells. May regulate signaling cascades through ubiquitination. Adapter that mediates the interaction between TRAF6 and CYLD (By similarity). May be involved in cell differentiation, apoptosis, immune response and regulation of K(+) channels. -
組織特異性
Ubiquitously expressed. -
関連疾患
Defects in SQSTM1 are a cause of Paget disease of bone (PDB) [MIM:602080]. PDB is a metabolic bone disease affecting the axial skeleton and characterized by focal areas of increased and disorganized bone turn-over due to activated osteoclasts. Manifestations of the disease include bone pain, deformity, pathological fractures, deafness, neurological complications and increased risk of osteosarcoma. PDB is a chronic disease affecting 2 to 3% of the population above the age of 40 years. -
配列類似性
Contains 1 OPR domain.
Contains 1 UBA domain.
Contains 1 ZZ-type zinc finger. -
ドメイン
The UBA domain binds specifically 'Lys-63'-linked polyubiquitin chains of polyubiquitinated substrates. Mediates the interaction with TRIM55.
The OPR domain mediates homooligomerization and interactions with PRKCZ, PRKCI, MAP2K5 and NBR1.
The ZZ-type zinc finger mediates the interaction with RIPK1. -
翻訳後修飾
Phosphorylated. May be phosphorylated by PRKCZ (By similarity). Phosphorylated in vitro by TTN. -
細胞内局在
Cytoplasm. Late endosome. Nucleus. Sarcomere (By similarity). In cardiac muscles localizes to the sarcomeric band (By similarity). Localizes to late endosomes. May also localize to the nucleus. Accumulates in neurofibrillary tangles and in Lewy bodies of neurons from individuals with Alzheimer and Parkinson disease respectively. Enriched in Rosenthal fibers of pilocytic astrocytoma. In liver cells, accumulates in Mallory bodies associated with alcoholic hepatitis, Wilson disease, indian childhood cirrhosis and in hyaline bodies associated with hepatocellular carcinoma. - Information by UniProt
関連製品
-
KO cell lysates
-
Related Products
アプリケーション
The Abpromise guarantee
Abpromise保証は、 次のテスト済みアプリケーションにおけるab266871の使用に適用されます
アプリケーションノートには、推奨の開始希釈率がありますが、適切な希釈率につきましてはご検討ください。
アプリケーション | Abreviews | 特記事項 |
---|---|---|
WB |
Use at an assay dependent concentration. Predicted molecular weight: 47 kDa.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
特記事項 |
---|
WB
Use at an assay dependent concentration. Predicted molecular weight: 47 kDa. Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
画像
-
All lanes : Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker (ab109012)
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab109012 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.
ab109012 Anti-SQSTM1 / p62 antibody [EPR4844] - Autophagosome Marker was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in knockout cell line ab266871 (knockout cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab109012 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
All lanes : Anti-SQSTM1 / p62 antibody [EPR18351] (ab207305)
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : SQSTM1 knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 47 kDa
Observed band size: 55 kDa why is the actual band size different from the predicted?Lanes 1-4: Merged signal (red and green). Green - ab207305 observed at 55 kDa. Red - loading control ab8245 observed at 36 kDa.
ab207305 Anti-SQSTM1 / p62 antibody [EPR18351] was shown to specifically react with SQSTM1 / p62 in wild-type HCT116 cells. The band observed in knockout cell line ab266871 (knockout cell lysate ab257052) lane below 55 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and SQSTM1 / p62 knockout samples were subjected to SDS-PAGE. ab207305 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
Homozygous: 49 bp deletion in exon4
-
Representative images of SQSTM1 knockout HCT116 cells, low and high confluency examples (top left and right respectively) and wild-type HCT116 cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.
プロトコール
データシートおよび資料
-
SDS download
-
Datasheet download
参考文献 (0)
ab266871 は論文での使用が確認できていません。