人RXRA knockout HCT116 cell line (ab273708)
概述
-
产品名称
人RXRA knockout HCT116 cell line -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 1 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
经测试应用
适用于: WB, ICC/IFmore details -
Biosafety level
1 -
常规说明
Recommended control: Human wild-type HCT116 cell line (ab273730). 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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.
性能
-
Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Colon -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
Antibiotic resistance
Puromycin 1.00µg/ml -
Mycoplasma free
Yes -
存放说明
Shipped on Dry Ice. Store in liquid nitrogen. -
存储溶液
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
研究领域
靶标
-
功能
Receptor for retinoic acid. Retinoic acid receptors bind as heterodimers to their target response elements in response to their ligands, all-trans or 9-cis retinoic acid, and regulate gene expression in various biological processes. The RAR/RXR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. The high affinity ligand for RXRs is 9-cis retinoic acid. RXRA serves as a common heterodimeric partner for a number of nuclear receptors. The RXR/RAR heterodimers bind to the retinoic acid response elements (RARE) composed of tandem 5'-AGGTCA-3' sites known as DR1-DR5. In the absence of ligand, the RXR-RAR heterodimers associate with a multiprotein complex containing transcription corepressors that induce histone acetylation, chromatin condensation and transcriptional suppression. On ligand binding, the corepressors dissociate from the receptors and associate with the coactivators leading to transcriptional activation. The RXRA/PPARA heterodimer is required for PPARA transcriptional activity on fatty acid oxidation genes such as ACOX1 and the P450 system genes. -
组织特异性
Highly expressed in liver, also found in lung, kidney and heart. -
序列相似性
Belongs to the nuclear hormone receptor family. NR2 subfamily.
Contains 1 nuclear receptor DNA-binding domain. -
结构域
Composed of three domains: a modulating N-terminal domain (AF1 domain), a DNA-binding domain and a C-terminal ligand-binding domain (AF2 domain). -
翻译后修饰
Phosphorylated on serine and threonine residues mainly in the N-terminal modulating domain. Constiutively phosphorylated on Ser-21 in the presence or absence of ligand. Under stress conditions, hyperphosphorylated by activated JNK on Ser-56, Ser-70, Thr-82 and Ser-260 (By similarity). Phosphorylated on Ser-27, in vitro, by PKA. This phosphorylation is required for repression of cAMP-mediated transcriptional activity of RARA.
Sumoylation negatively regulates transcriptional activity. Desumoylated specifically by SENP6. -
细胞定位
Nucleus. - Information by UniProt
相关产品
-
KO cell lysates
-
Related Products
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab273708于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度；实际最佳的稀释度/浓度应由使用者检定。
| 应用 | Ab评论 | 说明 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa.
|
|
| ICC/IF |
Use at an assay dependent concentration.
|
| 说明 |
|---|
|
WB
Use at an assay dependent concentration. Predicted molecular weight: 51 kDa. |
|
ICC/IF
Use at an assay dependent concentration. |
图片
-
Western blot - Human RXRA knockout HCT116 cell line (ab273708)All lanes : Anti-Retinoid X Receptor alpha/RXRA antibody [EPR7106] (ab125001) at 1/500 dilution
Lane 1 : Wild-type HCT116 lysate
Lane 2 : RXRA knock-out HCT116 lysate
Lysates/proteins at 20 µg per lane.
Predicted band size: 51 kDa
Observed band size: 51 kDa
ab125001 was shown to react with RXRA in wild-type HCT116 cells in Western blot with loss of signal observed in RXRA knockout cell line ab273708. Wild-type HCT116 and RXRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab125001 overnight at 4°C at a 1/500 dilution. Blots were incubated with secondary antibodies at 0.2µg/mL before imaging.
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies. -
Western blot - Human RXRA knockout HCT116 cell line (ab273708)All lanes : Anti-Retinoid X Receptor alpha/RXRA antibody [EPR7106] (ab125001) at 1/1000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : RXRA knockout HCT116 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 51 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab125001 observed at 53 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab125001 was shown to react with Anti-Retinoid X Receptor alpha in wild-type HCT 116 cells in western blot with loss of signal observed in RXRA knockout cell line ab273708 (RXRA knockout cell lysate ab275245). Wild-type and RXRA knockout HCT 116 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab125001 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
-
Immunocytochemistry/ Immunofluorescence - Human RXRA knockout HCT116 cell line (ab273708)ab125001 staining Retinoid X Receptor alpha in wild-type HCT116 cells (top panel) and RXRA knockout HCT116 cells (bottom panel) (ab273708). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab125001 at 0.2μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Sanger Sequencing - Human RXRA knockout HCT116 cell line (ab273708)Allele-1: 1 bp insertion in exon 1.
实验方案
数据表及文件
-
SDS download
-
Datasheet download
文献 (0)
ab273708 尚未被引用在任何文献中。