Human ACTA2 knockout HeLa cell line (ab264014)
Overview
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Product name
Human ACTA2 knockout HeLa cell line
See all alpha smooth muscle Actin lysates -
Parental Cell Line
HeLa -
Organism
Human -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: ICC, WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab271142). 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: DMEM (High Glucose) + 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
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
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
Actins are highly conserved proteins that are involved in various types of cell motility and are ubiquitously expressed in all eukaryotic cells. -
Involvement in disease
Defects in ACTA2 are the cause of aortic aneurysm familial thoracic type 6 (AAT6) [MIM:611788]. AATs are characterized by permanent dilation of the thoracic aorta usually due to degenerative changes in the aortic wall. They are primarily associated with a characteristic histologic appearance known as 'medial necrosis' or 'Erdheim cystic medial necrosis' in which there is degeneration and fragmentation of elastic fibers, loss of smooth muscle cells, and an accumulation of basophilic ground substance. -
Sequence similarities
Belongs to the actin family. -
Cellular localization
Cytoplasm > cytoskeleton. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
- Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964)
- Anti-alpha smooth muscle Actin antibody [SP171] (ab150301)
- Alexa Fluor® 488 Anti-alpha smooth muscle Actin antibody [EPR5368] (ab202295)
- Alexa Fluor® 555 Anti-alpha smooth muscle Actin antibody [EPR5368] (ab202509)
- Anti-FHL2 antibody [EPR17860-23] (ab202586)
- HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696)
- Anti-alpha smooth muscle Actin antibody [EPR5368] - BSA and Azide free (ab220795)
- Anti-Lin28B antibody [EPR18717] - BSA and Azide free (ab240326)
- Anti-alpha smooth muscle Actin antibody [1A4] - BSA and Azide free (ab240654)
- Anti-alpha smooth muscle Actin antibody [SP171] - BSA and Azide free (ab242395)
- Anti-FHL2 antibody [EPR17860-23] - BSA and Azide free (ab251380)
- Anti-alpha smooth muscle Actin antibody [1A4] (ab7817)
- FITC Anti-alpha smooth muscle Actin antibody [1A4] (ab8211)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab264014 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 |
|---|---|---|
| ICC |
Use at an assay dependent concentration.
|
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| WB |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
ICC
Use at an assay dependent concentration. |
|
WB
Use at an assay dependent concentration. |
Images
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Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)
ab202509 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab202509 at 1/200 dilution and ab195884 (Rat monoclonal to Tubulin - Alexa Fluor® 647) at 1/100 dilution overnight at 4oC. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Western blot - Human ACTA2 knockout HeLa cell line (ab264014)All lanes : HRP Anti-alpha smooth muscle Actin antibody [1A4] (ab203696) at 1/5000 dilution
Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Exposure time: 20 secondsab203696 was shown to react with alpha smooth muscle Actin (HRP) in wild-type HeLa cells in western blot. Loss of signal was observed when ACTA2 knockout cell line ab264014 (knockout cell lysate ab264499) was used. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab203696 overnight at 4°C at a 1 in 5000 dilution and ab184095 (Mouse Anti-GAPDH antibody [mAbcam 9484] - Alexa Fluor® 680) at a 1 in 1000 dilution. Blots were developed with Optiblot ECL reagent (ab133456) and imaged.
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Western blot - Human ACTA2 knockout HeLa cell line (ab264014)All lanes : Anti-alpha smooth muscle Actin antibody [SP171] (ab150301) at 1/130 dilution
Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 2: Merged signal (red and green). Green - ab150301 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab150301 was shown to react with alpha smooth muscle Actin in wild-type HeLa cells in western blot Loss of signal was observed when ACTA2 knockout cell line ab264014 (knockout cell lysate ab264499) was used. Wild-type HeLa and ACTA2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab150301 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 130 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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Western blot - Human ACTA2 knockout HeLa cell line (ab264014)All lanes : Anti-alpha smooth muscle Actin antibody [EPR5368] (ab124964) at 1/10000 dilution
Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.Lanes 1 - 2: Merged signal (red and green). Green - ab124964 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.
ab124964 was shown to react with alpha smooth muscle Actin in wild-type HeLa cells in western blot Loss of signal was observed when ACTA2 knockout cell line ab264014 (knockout cell lysate ab264499) was used. Wild-type HeLa and ACTA2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab124964 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 10000 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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Western blot - Human ACTA2 knockout HeLa cell line (ab264014)All lanes : Anti-alpha smooth muscle Actin antibody [1A4] (ab7817) at 1/131.58 dilution
Lane 1 : Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : ACTA2 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Performed under reducing conditions.Lanes 1 - 2: Merged signal (red and green). Green - ab7817 observed at 42 kDa. Red - loading control, ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.
ab7817 was shown to react with alpha smooth muscle Actin in wild-type HeLa cells in western blot Loss of signal was observed when ACTA2 knockout cell line ab264014 (knockout cell lysate ab264499) was used. Wild-type HeLa and ACTA2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab7817 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4°C at a 1 in 131.58 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging
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Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab202295 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab202295 at 1/500 dilution and ab195884 (Rat monoclonal to Tubulin - Alexa Fluor® 647) at 1/100 dilution overnight at 4oC. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab150301 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab150301 at 5 ug/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 ug/ml (shown in green) and a goat secondary antibody to mouse IgG (AAlexa Fluor® 594) (ab150120) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab124964 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab124964 at 1/500 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 ug/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab8211 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab8211 at 5 ug/ml concentration and ab195884 (Rat monoclonal to Tubulin - Alexa Fluor® 647) at 1/100 dilution overnight at 4oC. Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab7817 staining alpha smooth muscle Actin in wild-type HeLa cells (top panel) and ACTA2 knockout HeLa cells (ab264014) (bottom panel). The cells were fixed with 100% methanol (5 min) then 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 ab7817 at 5ug/ml concentration and ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4oC followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117) at 2 ug/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab202586 staining FHL2 in wild-type U-2 OS cells (top panel) and FHL2 knockout U-2 OS cells (ab264014) (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 ab202586 at 1/100 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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Immunocytochemistry/ Immunofluorescence - Human ACTA2 knockout HeLa cell line (ab264014)ab202586 staining FHL2 in wild-type U-2 OS cells (top panel) and FHL2 knockout U-2 OS cells (ab264014) (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 ab202586 at 1/100 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)
ab264014 has not yet been referenced specifically in any publications.