Human ALDH1A1 knockout A549 cell line (ab261864)
Overview
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Product name
Human ALDH1A1 knockout A549 cell line
See all ALDH1A1 lysates -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 8 bp deletion; Frameshift: 98% -
Passage number
<20 -
Knockout validation
Next Generation Sequencing (NGS), Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type A549 cell line (ab259777). 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:Hams F12 + 5% 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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Lung -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
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
Binds free retinal and cellular retinol-binding protein-bound retinal. Can convert/oxidize retinaldehyde to retinoic acid. -
Pathway
Cofactor metabolism; retinol metabolism. -
Sequence similarities
Belongs to the aldehyde dehydrogenase family. -
Cellular localization
Cytoplasm. - 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 ab261864 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 |
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WB |
Use at an assay dependent concentration.
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Notes |
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WB
Use at an assay dependent concentration.
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Images
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All lanes : Anti-ALDH1A1 antibody [EP1933Y] - C-terminal (ab52492) at 1/1000 dilution
Lane 1 : Wild-type A549 cell lysate
Lane 2 : ALDH1A1 knockout A549 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : MCF7 cell lysate
Lysates/proteins at 20 µg per lane.Western blot: Anti-ALDH1A1 antibody [EP1933Y] (ab52492) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab52492 was shown to bind specifically to ALDH1A1. A band was observed at 55 kDa in wild-type A549 cell lysates with no signal observed at this size in ALDH1A1 knockout cell line. To generate this image, wild-type and ALDH1A1 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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All lanes : Anti-ALDH1A1 antibody [ALDH1A1/1381] (ab219303) at 1 µg/ml
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : ALDH1A1 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Observed band size: 55 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab219303 observed at 55 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab219303 was shown to specifically react with ALDH1A1 in wild-type A549 cells as signal was lost in ALDH1A1 knockout cell line ab261864 (knockout cell lysate ab261673). Wild-type and ALDH1A1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab219303 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.
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Knockout achieved by CRISPR/Cas9; X = 8 bp deletion; Frameshift: 98%
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261864 has not yet been referenced specifically in any publications.