FMR1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
A549
Human
Lung
Liquid
Western blot
FMR1_HUMAN, FMRP, FRAXA, Fmr1 gene, Fragile X mental retardation 1, Fragile X mental retardation 1 protein, Fragile X mental retardation protein, Fragile X mental retardation protein 1, Fragile X mental retardation syndrome-related protein 1, MGC87458, POF, POF1, Protein FMR-1, fragile X mental retardation, autosomal homolog 1, wu:fb16f11, wu:fd18c10, zgc:66226
FMR1 KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided.
A549
Human
Lung
Liquid
Western blot
Carcinoma
FMR1
Knockout
CRISPR technology
Western blot
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
EU: 1 US: 1
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
-196°C
-196°C
Although we aim to provide customers with a homozygous clone, feasibility will be dependent on the biology of the protein. Should only heterozygous edits be achieved, you will be notified of the outcome and be asked to confirm whether the cell line is acceptable. All clones will be accompanied with DNA sequencing data, and the mutation description.
Recommended control: Human wild-type A549 cell line (Human wild-type A549 cell line ab288558). 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.
We will provide viable cells that proliferate on revival.
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 are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
Western blot: Anti-FMRP antibody Anti-FMRP antibody ab109741 staining at 1 µg/mL, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 75 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in FMR1 knockout U-87 MG cell line. To generate this image, 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 Donkey anti-Goat 800CW & Donkey anti-Mouse 680RD at 1/20,000 dilution.
All lanes: Western blot - Anti-FMRP antibody (Anti-FMRP antibody ab109741) at 1 µg/mL
Lane 1: Wild-type U-87 MG at 20 µg
Lane 2: Western blot - Human FMR1 knockout U-87 MG cell line (Human FMR1 knockout U-87 MG cell line ab306664) at 20 µg
Lane 3: Wild-type A549 at 20 µg
Lane 4: Western blot - Human FMR1 knockout A549 cell line (ab288956) at 20 µg
All lanes: Donkey anti-Goat 800CW & Donkey anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 75 kDa
Western blot: Anti-FMRP antibody Anti-FMRP antibody ab17722 staining at 1 µg/mL, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 75 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in FMR1 knockout U-87 MG cell line. To generate this image, 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-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.
All lanes: Western blot - Anti-FMRP antibody (Anti-FMRP antibody ab17722) at 1 µg/mL
Lane 1: Wild-type U-87 MG at 20 µg
Lane 2: Western blot - Human FMR1 knockout U-87 MG cell line (Human FMR1 knockout U-87 MG cell line ab306664) at 20 µg
Lane 3: Wild-type A549 at 20 µg
Lane 4: Western blot - Human FMR1 knockout A549 cell line (ab288956) at 20 µg
All lanes: Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 75 kDa
False colour image of Western blot: Anti-FMRP antibody staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-FMRP antibody ab17722 was shown to bind specifically to FMRP. A band was observed at 77 kDa in wild-type A549 cell lysates with no signal observed at this size in Fmr1 knockout cell line. To generate this image, wild-type and Fmr1 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-FMRP antibody (Anti-FMRP antibody ab17722) at 1 µg
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: Fmr1 knockout A549 cell lysate at 20 µg
Lane 3: HeLa cell lysate at 20 µg
Lane 4: Human Brain cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 77 kDa
False colour image of Western blot: Anti-FMRP antibody [EPR23852-90] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-FMRP antibody [EPR23852-90] ab259335 was shown to bind specifically to FMRP. A band was observed at 70-77 kDa in wild-type A549 cell lysates with no signal observed at this size in Fmr1 knockout cell line ab288956. To generate this image, wild-type and Fmr1 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes: Western blot - Anti-FMRP antibody [EPR23852-90] (Anti-FMRP antibody [EPR23852-90] ab259335) at 1/1000 dilution
Lane 1: Wild-type A549 cell lysate at 20 µg
Lane 2: Fmr1 knockout A549 cell lysate at 20 µg
Lane 3: HeLa cell lysate at 20 µg
Lane 4: Human Brain cell lysate at 20 µg
Performed under reducing conditions.
Observed band size: 70-77 kDa
40 bp deletion after the Thr236 of the WT protein
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