Human CHEK1 heterozygous knockout A549 cell line (ab276102)
製品の概要
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製品名
Human CHEK1 heterozygous knockout A549 cell line -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Heterozygous: 46 bp deletion in exon 3 and wild-type. -
Passage number
<20 -
Knockout validation
Sanger Sequencing -
Biosafety level
1 -
特記事項
Recommended control: Human wild-type A549 cell line (ab275463). 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: F-12K + 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 2x103-1x104 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 6x104 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.
- Do not exceed 7x104 cells/cm2.
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 -
Viability
~50% -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
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
画像
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Western blot - Human CHEK1 heterozygous knockout A549 cell line (ab276102)All lanes : Anti-CHEK1 antibody at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : CHEK1 knockout A549 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : MOLT-4 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 57 kDa why is the actual band size different from the predicted?Anti-CHEK1 antibody [2G1D5] staining at 1/1000 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, this antibody was shown to bind specifically to CHEK1. A band was observed at 57 kDa in wild-type A549 cell lysates with a reduction in signal observed at this size in CHEK1 heterozygous knockout cell line. To generate this image, wild-type and CHEK1 heterozygous knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.
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Western blot - Human CHEK1 heterozygous knockout A549 cell line (ab276102)All lanes : Anti-Chk1 antibody [EP691Y] (ab40866) at 1/10000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : CHEK1 knockout A549 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : MOLT-4 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 57 kDa why is the actual band size different from the predicted?Anti-CHEK1 antibody [EP691Y] (ab40866) 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, ab40866 was shown to bind specifically to CHEK1. A band was observed at 57 kDa in wild-type A549 cell lysates with a reduction in signal observed at this size in CHEK1 heterozygous knockout cell line. To generate this image, wild-type and CHEK1 heterozygous knockout A549 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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Western blot - Human CHEK1 heterozygous knockout A549 cell line (ab276102)All lanes : Anti-Chk1 antibody [E250] (ab32531) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : CHEK1 knockout A549 cell lysate
Lane 3 : A431 cell lysate
Lane 4 : MOLT-4 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 57 kDa why is the actual band size different from the predicted?Anti-CHEK1 antibody [E250] (ab32531) 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, ab32531 was shown to bind specifically to CHEK1. A band was observed at 57 kDa in wild-type A549 cell lysates with a reduction in signal observed at this size in CHEK1 heterozygous knockout cell line. To generate this image, wild-type and CHEK1 heterozygous knockout A549 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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Sanger Sequencing - Human CHEK1 knockout A549 cell line (ab276102)Sanger Sequencing - Human CHEK1 heterozygous knockout A549 cell line (ab276102).
47 bp deletion in exon 3 and wild type.
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)
ab276102 は論文での使用が確認できていません。