Human S100A4 knockout A549 cell line (ab261865)
Overview
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Product name
Human S100A4 knockout A549 cell line
See all S100A4 lysates -
Parental Cell Line
A549 -
Organism
Human -
Mutation description
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift = 95% -
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, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and 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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Tissue specificity
Ubiquitously expressed. -
Sequence similarities
Belongs to the S-100 family.
Contains 2 EF-hand domains. - 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 ab261865 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.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. |
Images
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Western blot - Human S100A4 knockout A549 cell line (ab261865)All lanes : Anti-S100A4 antibody [EPR2761(2)] (ab124805) at 1/1000 dilution
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : S100A4 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : A-375 (Human malignant melanoma 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: 12 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab124805 observed at 12 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab124805 was shown to react with S100A4 in wild-type A549 cells in Western blot with loss of signal observed in S100A4 knockout cell line ab261865 (knockout cell lysate ab261674). Wild-type A549 and S100A4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab124805 and ab8245 (Mouse anti-GAPDH antibody [6C5]) 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 h at room temperature before imaging.
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Western blot - Human S100A4 knockout A549 cell line (ab261865)All lanes : Anti-S100A4 antibody (ab41532) at 1/250 dilution
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : S100A4 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : A-375 (Human malignant melanoma cell line) whole cell lysate, 20 ug
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: 12 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab41532 observed at 12 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab41532 was shown to recognize S100A4 in wild-type A549 cells as signal was lost at the expected MW in S100A4 knockout cell line ab261865 (knockout cell lysate ab261674). Additional cross-reactive bands were observed in the wild-type and knockout samples. Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. Ab41532 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/250 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.
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Western blot - Human S100A4 knockout A549 cell line (ab261865)All lanes : Anti-S100A4 antibody [S100A4/1482] (ab218512) at 1 µg/ml
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : Human S100A4 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : A-375 (Human malignant melanoma 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.
Observed band size: 12 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab218512 observed at 12 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab218512 was shown to specifically react with S100A4 in wild-type A549 cells as signal was lost in S100A4 knockout cell line ab261865 (knockout cell lysate ab261674). Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. Ab218512 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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Western blot - Human S100A4 knockout A549 cell line (ab261865)All lanes : Anti-S100A4 antibody [S100A4/1481] (ab218511) at 1 µg/ml
Lane 1 : Wild-type A549 (Human lung carcinoma cell line) whole cell lysate
Lane 2 : S100A4 knockout A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : A-375 (Human malignant melanoma 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: 12 kDa why is the actual band size different from the predicted?Lanes 1 - 4: Merged signal (red and green). Green - ab218511 observed at 12 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab218511 was shown to specifically react with S100A4 in wild-type A549 cells as signal was lost in S100A4 knockout cell line ab261865 (knockout cell lysate ab261674). Wild-type and S100A4 knockout samples were subjected to SDS-PAGE. Ab218511 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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Next Generation Sequencing - Human S100A4 knockout A549 cell line (ab261865)Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift = 95%
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab261865 has not yet been referenced specifically in any publications.