Human RIPK1 knockout THP-1 cell line (ab276121)
製品の概要
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製品名
Human RIPK1 knockout THP-1 cell line -
Parental Cell Line
THP-1 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 77 bp deletion in exon 3 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
アプリケーション
適用あり: Sanger Sequencing, WBmore details -
Biosafety level
1 -
特記事項
Recommended control: Human wild-type THP-1 cell line (ab275477). 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: RPMI + 10% FBS + 0.05 mM β-mercaptoethanol
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 2-4x105 cells/mL. 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.
5. THP-1 cells recover slowly from cryopreservation and therefore may not be ready for subculture for a number of days. Cells should be left as much as possible over this time and only subcultured when the cell density reaches 8x105 cells/mL.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- Cells should be seeded at 2-4x105 cells/mL and subcultured when they have reached 8x105 cells/mL. It is not recommended to allow the cell density to exceed 1x106 cells/mL.
- 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
Suspension -
Tissue
Blood -
Cell type
acute monocytic leukemia -
Disease
Acute Monocytic Leukemia -
Gender
Male -
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
Essential adapter molecule for the activation of NF-kappa-B. Following different upstream signals (binding of inflammatory cytokines, stimulation of pathogen recognition receptors, or DNA damage), particular RIPK1-containing complexes are formed, initiating a limited number of cellular responses. Upon TNFA stimulation RIPK1 is recruited to a TRADD-TRAF complex initiated by TNFR1 trimerization. There, it is ubiquitinated via 'Lys-63'-link chains, inducing its association with the IKK complex, and its activation through NEMO binding of polyubiquitin chains. -
配列類似性
Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family.
Contains 1 death domain.
Contains 1 protein kinase domain. -
翻訳後修飾
Proteolytically cleaved by caspase-8 during TNF-induced apoptosis. Cleavage abolishes NF-kappa-B activation and enhances pro-apototic signaling through the TRADD-FADD interaction.
Autophosphorylated on serine and threonine residues.
Ubiquitinated by 'Lys-11'-, 'Lys-48'-, 'Lys-63'- and linear-linked type ubiquitin. Polyubiquitination with 'Lys-63'-linked chains by TRAF2 induces association with the IKK complex. Deubiquitination of 'Lys-63'-linked chains and polyubiquitination with 'Lys-48'-linked chains by TNFAIP3 leads to RIPK1 proteasomal degradation and consequently to the termination of the TNF- or Linear polyubiquitinated; the head-to-tail polyubiquitination is mediated by the LUBAC complex. LPS-mediated activation of NF-kappa-B. Also ubiquitinated with 'Lys-11'-linked chains. -
細胞内局在
Cytoplasm. - Information by UniProt
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ab276121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 |
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Sanger Sequencing |
Use at an assay dependent concentration.
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 75 kDa.
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特記事項 |
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Sanger Sequencing
Use at an assay dependent concentration. |
WB
Use at an assay dependent concentration. Predicted molecular weight: 75 kDa. |
画像
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All lanes : Anti-RIP antibody [EPR4689] (ab125072) at 1/1000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : RIPK1 knockout THP-1 cell lysate
Lane 3 : Raji cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 75 kDa
Observed band size: 75 kDaWestern blot: Anti-RIPK1 antibody [EPR4689] (ab125072) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab125072 was shown to bind specifically to RIPK1. A band was observed at 75 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line. To generate this image, wild-type and RIPK1 knockout THP-1 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-RIP antibody [EPR4689-100] (ab178420) at 1/1000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : RIPK1 knockout THP-1 cell lysate
Lane 3 : Raji cell lysate
Lane 4 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 75 kDa
Observed band size: 75 kDaWestern blot: Anti-RIPK1 antibody [EPR4689-100] (ab178420) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab178420 was shown to bind specifically to RIPK1. A band was observed at 75 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line. To generate this image, wild-type and RIPK1 knockout THP-1 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
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All lanes : Anti-RIP antibody [EPR24883-85] (ab300617) at 1/1000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : RIPK1 knockout THP-1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 75 kDa
Observed band size: 75 kDaWestern blot: Anti-RIPK1 antibody [EPR24883-85] (ab300617) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab300617 was shown to bind specifically to RIPK1. A band was observed at 75 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284221). To generate this image, wild-type and RIPK1 knockout THP-1 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
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All lanes : Anti-RIP antibody [EPR4689-100] (ab178420) at 1/1000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : RIPK1 knockout THP-1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 75 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-RIP antibody [EPR4689-100] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab178420 was shown to bind specifically to RIP. A band was observed at 76 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284210). To generate this image, wild-type and RIPK1 knockout THP-1 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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All lanes : Anti-RIP antibody [EPR4689] (ab125072) at 1/1000 dilution
Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : RIPK1 knockout THP-1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Raji cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 75 kDa
Observed band size: 76 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-RIP antibody [EPR4689] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab125072 was shown to bind specifically to RIP. A band was observed at 76 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284210). To generate this image, wild-type and RIPK1 knockout THP-1 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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77bp deletion in exon 3.
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)
ab276121 は論文での使用が確認できていません。