AB266846

Human ADAR (ADAR1) knockout HEK-293T cell line

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ADAR KO cell line available to order. KO validated by Western blot. Free of charge wild type control available. Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 2 and 1 bp insertion in exon 2. To order both knockout and wild-type control cells: select 2 x 1000000Cells/vial. To order only knockout cells: select 1000000Cells/vial.

別名を表示する

136 kDa double-stranded RNA-binding protein, ADAR, AGS6, AV242451, Adenosine deaminase RNA specific, Adenosine deaminase RNA specific 1, Adenosine deaminase acting on RNA 1 A, Adenosine deaminase that act on RNA, DRADA, DSRAD_HUMAN, Double-stranded RNA-specific adenosine deaminase, Double-stranded RNA-specific editase Adar, Dsh, EC 3.5.4.-, G1P1, Ifi4 protein, Interferon induced protein 4, Interferon-inducible protein 4, K88DSRBP, P136, Pre-mRNA adenosine deaminase, RNA adenosine deaminase 1, RNA-editing deaminase 1, RNA-editing enzyme 1, dsRNA adenosine deaminase, mZaADAR

6 Images

Immunocytochemistry/ Immunofluorescence - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized ADAR KO HEK293T (ab266846) cells labelling ADAR1 with ab307585 at 1/500 (1.08 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing increased nuclear and cytoplasmic staining in parental HEK293T cells treated with IFN alpha 1 (human) (10 ng/ml) for 16 hours, and no staining in treated ADAR KO HEK293T cells. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Lab

Western blot - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Lanes 1-3 : Merged signal (red and green). Green - ab126745 observed at 130 kDa. Red - loading control ab8245 observed at 36 kDa.

ab126745 Anti-ADAR1 antibody [EPR7033] was shown to specifically react with ADAR1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266846 (knockout cell lysate ab257131) was used. Wild-type and ADAR1 knockout samples were subjected to SDS-PAGE. ab126745 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° 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:

Western blot - Anti-ADAR1 antibody [EPR7033] (<a href='/products/primary-antibodies/adar1-antibody-epr7033-ab126745'>ab126745</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

ADAR knockout HEK293T cell lysate at 20 µg

Lane 2:

Western blot - Human ADAR (ADAR1) knockout HEK-293T cell line (ab266846)

Lane 3:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 136 kDa,19 kDa,29 kDa,35 kDa,36 kDa

Observed band size: 130 kDa,20 kDa,29 kDa,35 kDa,36 kDa

false

Supplier Data

Western blot - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Performed under reducing conditions. In Western blot, ab307585 was shown to bind specifically to ADAR1. A band was observed at 150 kDa in wild-type HEK-293T cell lysates whereas no signal observed at this size in ADAR1 knockout cell line ab266846 (knockout cell lysate ab257131). Exposure time : 59 seconds

All lanes:

Western blot - Anti-ADAR1 antibody [EPR25431-60] (<a href='/products/primary-antibodies/adar1-antibody-epr25431-60-ab307585'>ab307585</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

ADAR1 knockout HEK-293T whole cell lysate at 20 µg

Lane 2:

Western blot - Human ADAR (ADAR1) knockout HEK-293T cell line (ab266846)

Lane 2:

Western blot - Human ADAR (ADAR1) knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-adar-adar1-knockout-hek-293t-cell-lysate-ab257131'>ab257131</a>)

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Ramos (human burkitts lymphoma b lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 150 kDa

false

Exposure time: 59s

Sanger Sequencing - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Sanger seq

Unknown

Sanger Sequencing - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Allele-2 : 1 bp insertion in exon 2.

Sanger seq

Lab

Sanger Sequencing - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Sequencing chromatogram displaying sequence edit in exon 2

Sanger seq

Unknown

Sanger Sequencing - Human ADAR (ADAR1) knockout HEK-293T cell line (AB266846)

Allele-1 : 17 bp deletion in exon2

Key facts

細胞タイプ

HEK-293T

生物種

Human

組織

Kidney

製品の状態

Liquid

form

ノックアウト検証方法

Sanger Sequencing,Western blot

ノックアウト変異

Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 2 and 1 bp insertion in exon 2

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Reactivity data

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製品の詳細

We will provide viable cells that proliferate on revival.

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.

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製品内容

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出荷温度及び保存条件

遺伝子名

ADAR

遺伝子編集のタイプ

Knockout

遺伝子編集の方法

CRISPR technology

ノックアウト検証方法

Sanger Sequencing, Western blot

出荷温度

Dry Ice

短期保存温度

-196°C

長期保存温度

-196°C

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取り扱い方法

初回取り扱いガイドライン

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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO₂. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. Cultures should be monitored daily.

継代培養ガイドライン

All seeding densities should be based on cell counts gained by established methods.
A guide seeding density of 2x10⁴ cells/cm² is recommended.
Cells should be passaged when they have achieved 80-90% confluence.

培養培地

DMEM (High Glucose) + 10% FBS

凍結保存培地

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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補足情報

This supplementary information is collated from multiple sources and compiled automatically.

ADAR1 also known as RNA-specific adenosine deaminase 1 or ADAR is an enzyme with a mass of approximately 150 kDa. This protein targets double-stranded RNA (dsRNA) and acts mechanistically to convert adenosine to inosine in pre-mRNA sequences a process known as A-to-I RNA editing. ADAR1 is expressed in numerous tissues with high levels in the brain liver and lungs. Its localization within cells can vary often found in both the nucleus and cytoplasm which influences its function.

Biological function summary

ADAR1 plays a role in the regulation of RNA molecules affecting their stability and translation. It is linked to the editosome complex working alongside other proteins to perform RNA editing tasks. By modifying the coding potential of mRNAs ADAR1 contributes to the diversity of proteomes and helps manage the responses to viral RNAs giving the immune system tools to recognize endogenous and exogenous RNA.

Pathways

ADAR1 is significant in the interferon signaling pathway and RNA processing pathways. It operates in coordination with proteins like PKR which is involved in the response to viral infections. ADAR1 ensures that the immune response is not directed against the self highlighting its role in the regulation of the innate immune system. These pathways are critical in maintaining homeostasis and preventing unchecked immune responses.

Mutations or dysregulation of ADAR1 have associations with autoimmune diseases like Aicardi-Goutieres syndrome and certain cancers. The enzyme's role in editing RNA makes it essential in preventing inappropriate immune attacks against the body's own cells highlighting its interaction with MDA5 another protein involved in immune regulation. Understanding ADAR1 and its related pathways may offer potential therapeutic targets for these conditions including exploration into ADAR1 inhibitors as interventions.

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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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製品プロトコール

Visit the General protocols
Visit the Troubleshooting

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ターゲットの情報

See full target information ADAR

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Human ADAR (ADAR1) knockout HEK-293T cell line