CD44 KO cell line available to order. KO validated by Immunocytochemistry, Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp deletion; Frameshift = 98.54%.
Images
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Next Generation Sequencing, Western blot
- Mutation description
- Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp deletion; Frameshift = 98.54%
Alternative names
BA-1, CD44 antigen, CD44 molecule, CD44 molecule (Indian blood group), CD44_HUMAN, CDW44 antigen, CDw44, CSPG8, Cell surface glycoprotein CD44, ECMR-III, Epican, Extracellular matrix receptor III, GP90 lymphocyte homing/adhesion receptor, HCELL, HSA, HUTCH-I, HUTCH1, Heparan sulfate proteoglycan, Hermes antigen, Hyaluronate receptor, IN, INLU-related p80 Glycoprotein, LHR, MC56, MDU2, MDU3, MGC10468, MIC4, MUTCH I, MUTCH1, PGP-1, Phagocytic glycoprotein 1, Phagocytic glycoprotein I, Soluble CD44, chondroitin sulfate proteoglycan 8, hematopoietic cell E- and L-selectin ligand, homing function and Indian blood group system
Recommended products
CD44 KO cell line available to order. KO validated by Immunocytochemistry, Next Generation Sequencing, Western blot. Free of charge wild type control provided. Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp deletion; Frameshift = 98.54%.
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Next Generation Sequencing, Western blot
- Mutation description
- Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp deletion; Frameshift = 98.54%
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- CD44
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Immunocytochemistry, Next Generation Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 2 US: 2
- Adherent/suspension
- Adherent
- Gender
- Female
Handling procedures
- 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 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.- 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.
- Cells should be passaged when they have achieved 80-90% confluence.
- Culture medium
- DMEM (High Glucose) + 10% FBS
- Cryopreservation medium
- Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
We will provide viable cells that proliferate on revival.
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.
Product promise
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.
14 product images
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - HRP Anti-CD44 antibody [EPR1013Y] ab194989 observed at 80 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.HRP Anti-CD44 antibody [EPR1013Y] ab194989 was shown to react with CD44 (HRP) in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with HRP Anti-CD44 antibody [EPR1013Y] ab194989 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4° at a 1 in 2500 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - HRP Anti-CD44 antibody [EPR1013Y] (HRP Anti-CD44 antibody [EPR1013Y] ab194989) at 1/2500 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
False colour image of Western blot: Anti-CD44 antibody [EPR1013Y] staining at 1/5000 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-CD44 antibody [EPR1013Y] ab51037 was shown to bind specifically to CD44. A band was observed at 75-80 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD44 knockout cell line ab262515 (knockout cell lysate Human CD44 knockout HeLa cell lysate ab263938). To generate this image wild-type and CD44 knockout HeLa 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°. 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-CD44 antibody [EPR1013Y] (Anti-CD44 antibody [EPR1013Y] ab51037) at 1/5000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: CD44 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 cell lysate at 20 µg
Lane 4: LNCaP cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 75-80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
False colour image of Western blot: Anti-CD44 antibody [C44Mab-5] staining at 1.226 μg/ml shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-CD44 antibody [C44Mab-5] ab264539 was shown to bind specifically to CD44. A band was observed at 75-80 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD44 knockout cell line ab262515 (knockout cell lysate Human CD44 knockout HeLa cell lysate ab263938). To generate this image wild-type and CD44 knockout HeLa 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°. 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
All lanes: Western blot - Anti-CD44 antibody [C44Mab-5] (Anti-CD44 antibody [C44Mab-5] ab264539) at 1.226 µg/mL
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: CD44 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 cell lysate at 20 µg
Lane 4: LNCaP cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 75-80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
False colour image of Western blot: Anti-CD44 antibody [EPR18668] staining at 1/1000 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-CD44 antibody [EPR18668] ab189524 was shown to bind specifically to CD44. A band was observed at 70-85 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD44 knockout cell line ab262515 (knockout cell lysate Human CD44 knockout HeLa cell lysate ab263938). To generate this image wild-type and CD44 knockout HeLa 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°. 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-CD44 antibody [EPR18668] (Anti-CD44 antibody [EPR18668] ab189524) at 1/1000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 70-85 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
False colour image of Western blot: Anti-CD44 antibody [SP37] staining at 1/1000 dilution shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution shown in red. In Western blot Anti-CD44 antibody [SP37] ab101531 was shown to bind specifically to CD44. A band was observed at 75-80 kDa in wild-type HeLa cell lysates with no signal observed at this size in CD44 knockout cell line ab262515 (knockout cell lysate Human CD44 knockout HeLa cell lysate ab263938). To generate this image wild-type and CD44 knockout HeLa 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°. 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.
All lanes: Western blot - Anti-CD44 antibody [SP37] (Anti-CD44 antibody [SP37] ab101531) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: CD44 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 cell lysate at 20 µg
Lane 4: LNCaP cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 75-80 kDa
Immunocytochemistry/ Immunofluorescence - Human CD44 knockout HeLa cell line (ab262515)
Anti-CD44 antibody [Hermes-3] ab254530 staining CD44 in wild-type HeLa cells (top panel) and CD44 knockout HeLa cells (ab262515) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Tween 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 Anti-CD44 antibody [Hermes-3] ab254530 at 0.4μg/ml concentration and Anti-beta Tubulin antibody - Loading Control ab6046 (Rabbit polyclonal to beta 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 mouse IgG (Alexa Fluor® 488) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44 antibody [BLR038F] ab243894 observed at 80 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.Anti-CD44 antibody [BLR038F] ab243894 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44 antibody [BLR038F] ab243894 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4° 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44 antibody [BLR038F] (Anti-CD44 antibody [BLR038F] ab243894) at 1/1000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44 antibody [EPR18668] ab189524 observed at 80 kDa. Red - loading control Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.Anti-CD44 antibody [EPR18668] ab189524 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44 antibody [EPR18668] ab189524 and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4° 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44 antibody [EPR18668] (Anti-CD44 antibody [EPR18668] ab189524) at 1/1000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44 antibody [EPR1013Y] ab51037 observed at 80 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.Anti-CD44 antibody [EPR1013Y] ab51037 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44 antibody [EPR1013Y] ab51037 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4° at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44 antibody [EPR1013Y] (Anti-CD44 antibody [EPR1013Y] ab51037) at 1/5000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44 antibody [SP37] ab101531 observed at 80 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.Anti-CD44 antibody [SP37] ab101531 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44 antibody [SP37] ab101531 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4° 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44 antibody [SP37] (Anti-CD44 antibody [SP37] ab101531) at 1/1000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44 antibody [MEM-263] ab9524 observed at 80 kDa. Red - loading control Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.Anti-CD44 antibody [MEM-263] ab9524 was shown to react with CD44 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type and CD44 knockout HeLa cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44 antibody [MEM-263] ab9524 and Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4° at 2 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44 antibody [MEM-263] (Anti-CD44 antibody [MEM-263] ab9524) at 2 µg/mL
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 80 kDa
Next Generation Sequencing - Human CD44 knockout HeLa cell line (ab262515)
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion, 1 bp deletion; Frameshift = 98.54%
Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lanes 1 - 4: Merged signal (red and green). Green - Anti-CD44v6 antibody [VFF-7] ab30436 observed at 80 kDa. Red - loading control Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.Anti-CD44v6 antibody [VFF-7] ab30436 was shown to react with CD44v6 in wild-type HeLa cells in western blot. Loss of signal was observed when CD44 knockout sample was used. Wild-type HeLa and CD44 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with Anti-CD44v6 antibody [VFF-7] ab30436 and Anti-alpha Tubulin antibody [EP1332Y] - Loading Control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4° at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-CD44v6 antibody [VFF-7] (Anti-CD44v6 antibody [VFF-7] ab30436) at 1/1000 dilution
Lane 1: Wild-type HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: CD44 knockout HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2: Western blot - Human CD44 knockout HeLa cell line (ab262515)
Lane 3: A549 (Human lung carcinoma cell line) whole cell lysate at 20 µg
Lane 4: LNCaP (Human prostate cancer cell line) whole cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 81 kDa, 82 kDa
Observed band size: 80 kDa
Next Generation Sequencing - Human CD44 knockout HeLa cell line (ab262515)
1 bp deletion after Arg149 (allele 1) and 1 bp deletion after Asn148 (allele 2) of the WT protein
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