DUSP4 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9,109 bp deletion (allele 1) and 110 bp deletion (allele 2) in exon 2, CCDS6072.1.
A549
Human
Lung
Liquid
Next Generation Sequencing, Western blot
Knockout achieved by using CRISPR/Cas9,109 bp deletion (allele 1) and 110 bp deletion (allele 2) in exon 2, CCDS6072.1
DUSP4 KO cell line available to order. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9,109 bp deletion (allele 1) and 110 bp deletion (allele 2) in exon 2, CCDS6072.1.
A549
Human
Lung
Liquid
Next Generation Sequencing, Western blot
Knockout achieved by using CRISPR/Cas9,109 bp deletion (allele 1) and 110 bp deletion (allele 2) in exon 2, CCDS6072.1
Carcinoma
DUSP4
Knockout
CRISPR technology
Next Generation Sequencing, Western blot
D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, D8S1179, TPOX, FGA, D19S433, D2S1338, VWA
EU: 1 US: 1
~ 80%
Adherent
Male
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.
F-12K + 10% FBS
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Dry Ice
A few days
-196°C
-196°C
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Western blot: Anti-DUSP4 antibody [EPR19881] (Anti-DUSP4 antibody [EPR19881] ab216576) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (Anti-Calnexin antibody [CANX/1543] ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, Anti-DUSP4 antibody [EPR19881] ab216576 was shown to bind specifically to DUSP4. A band was observed at 40 kDa in wild-type A549 cell lysates with no signal observed at this size in DUSP4 knockout cell line. To generate this image, wild-type and DUSP4 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.
All lanes: Western blot - Anti-DUSP4 antibody [EPR19881] (Anti-DUSP4 antibody [EPR19881] ab216576) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lanes 2 - 3: DUSP4 knockout A549 cell lysate at 20 µg
Lane 4: DUSP4 knockout A549 H15 cell lysate at 20 µg
Lane 5: HCT 116 cell lysate at 20 µg
Lane 6: MOLT-4 cell lysate at 20 µg
All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Performed under reducing conditions.
Observed band size: 40 kDa
109 bp deletion (allele 1) and 110 bp deletion (allele 2) in exon 2, CCDS6072.1
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