Human FHL2 knockout U-2 OS cell line (ab262496)
Overview
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Product name
Human FHL2 knockout U-2 OS cell line
See all FHL2 lysates -
Parental Cell Line
U-2 OS -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift = 96.21% -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type U-2 OS cell line (ab263976). 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 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Bone -
Cell type
epithelial -
Disease
Osteosarcoma -
Gender
Female -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
May function as a molecular transmitter linking various signaling pathways to transcriptional regulation. Negatively regulates the transcriptional repressor E4F1 and may function in cell growth. -
Tissue specificity
Expressed in skeletal muscle and heart. -
Sequence similarities
Contains 4 LIM zinc-binding domains. -
Domain
The third LIM zinc-binding mediates interaction with E4F1. -
Cellular localization
Cytoplasm. Nucleus. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab262496 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration.
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| ICC |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. |
|
ICC
Use at an assay dependent concentration. |
Images
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Western blot - Human FHL2 knockout U-2 OS cell line (ab262496)All lanes : Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution
Lane 1 : Wild-type U-2 OS cell lysate
Lane 2 : FHL2 knockout U-2 OS cell lysate
Lane 3 : HeLa cell lysate
Lysates/proteins at 40 µg per lane.Lanes 1 - 3: Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab202586 was shown to react with FHL2 in wild-type U-2 OS cells in western blot with loss of signal observed in FHL2 knockout sample. Wild-type and FHL2 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab202586 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] 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.
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Immunocytochemistry/ Immunofluorescence - Human FHL2 knockout U-2 OS cell line (ab262496)ab202584 staining FHL2 in wild-type U-2 OS cells (top panel) and FHL2 knockout U-2 OS cells (ab262496) (bottom panel). The cells were fixed with 4% PFA (10 min), 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 ab202584 at 1/200 dilution and ab7291 (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 pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
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Next Generation Sequencing - Human FHL2 knockout U-2 OS cell line (ab262496)Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift = 96.21%
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Western blot - Human FHL2 knockout U-2 OS cell line (ab262496)All lanes : Anti-FHL2 antibody [EPR17860-20] (ab202584) at 1/1000 dilution
Lane 1 : Wild-type U-2 OS cell lysate
Lane 2 : FHL2 knockout U-2 OS cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.Lanes 1 - 3: Merged signal (red and green). Green - ab202584 observed at 32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab202584 was shown to react with FHL2 in wild-type U-2 OS cells in western blot with loss of signal observed in FHL2 knockout cell line ab262496 (knockout cell lysate ab263928).Wild-type and FHL2 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab202584 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] 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.
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Immunocytochemistry/ Immunofluorescence - Human FHL2 knockout U-2 OS cell line (ab262496)ab202584 staining FHL2 in wild-type U-2 OS cells (top panel) and FHL2 knockout U-2 OS cells (ab262496) (bottom panel). The cells were fixed with 100% methanol (5 min), 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 ab202584 at 1/200 dilution and ab7291 (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 pseudo color red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab262496 has not yet been referenced specifically in any publications.