Human EPCAM knockout A-431 cell line
- Advanced Validation
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- Flow Cyt
Lab
Flow Cytometry - Human EPCAM knockout A-431 cell line (AB261902)
Flow cytometry overlay histogram showing wild-type A431 (green line) and EPCAM knockout A431 cells stained with ab85987 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab85987) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was mouse IgG1κ (ab170190) used at the same concentration and conditions as the primary antibody (wild-type A431 - black line; EPCAM knockout A431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab187372 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-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 ab187372 at 0.1ug/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. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab187372 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-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 ab187372 at 0.5μg/ml concentration and 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) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (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).
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab85987 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-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 ab85987 at 0.5μg/ml concentration and 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) (ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (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).
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab223582 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% PBS-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 ab223582 at 1/5000 dilution and ab7291 (Mouse monoclonal to alpha 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 red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab223582 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-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 ab223582 at 0.1ug/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 (Alexa Fluor® 594) (ab150120) at 2 ug/ml (shown in red). Nuclear DNA was labelled in blue with DAPI. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.
- Flow Cyt
Lab
Flow Cytometry - Human EPCAM knockout A-431 cell line (AB261902)
Flow cytometry overlay histogram showing wild-type A431 (green line) and EPCAM knockout A431 cells stained with ab223582 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab223582) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.
The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.
Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type A431 - black line; EPCAM knockout A431 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).
Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human EPCAM knockout A-431 cell line (AB261902)
ab85987 staining EpCAM in wild-type A-431 cells (top panel) and EpCAM knockout A-431 cells (ab261902) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then permeabilized with 0.1% PBS-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 ab85987 at 0.1ug/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. This antibody performed similarly using 100% methanol fixation. Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a single confocal section is shown.
- WB
Lab
Western blot - Human EPCAM knockout A-431 cell line (AB261902)
Lanes 1 - 3 : Merged signal (red and green). Green - ab223582 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab223582 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab223582 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4° at a 1 in 1000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-EpCAM antibody [EPR20532-225] (<a href='/en-us/products/primary-antibodies/epcam-antibody-epr20532-225-ab223582'>ab223582</a>) at 1/1000 dilution
Lane 1:
Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2:
EPCAM knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2:
Western blot - Human EPCAM knockout A-431 cell line (ab261902)
Lane 3:
HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Predicted band size: 35 kDa
Observed band size: 40 kDa
false
- WB
Lab
Western blot - Human EPCAM knockout A-431 cell line (AB261902)
Lanes 1 - 3 : Merged signal (red and green). Green - ab213500 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab213500 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab213500 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4° at a 1 in 1000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-EpCAM antibody [EPR20532-222] (<a href='/en-us/products/primary-antibodies/epcam-antibody-epr20532-222-ab213500'>ab213500</a>) at 1/1000 dilution
Lane 1:
Wild-type A431 whole cell lysate at 20 µg
Lane 2:
EPCAM knockout A431 whole cell lysate at 20 µg
Lane 2:
Western blot - Human EPCAM knockout A-431 cell line (ab261902)
Lane 3:
HeLa whole cell lysate at 20 µg
Predicted band size: 35 kDa
Observed band size: 35 kDa
false
- WB
Lab
Western blot - Human EPCAM knockout A-431 cell line (AB261902)
Lanes 1 - 4 : Merged signal (red and green). Green - ab32392 observed at 40 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.
ab32392 was shown to react with EpCAM in wild-type A431 cells in western blot with loss of signal observed in EpCAM knockout sample. Wild-type and EpCAM knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab32392 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4° at a 1 in 2000 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.
All lanes:
Western blot - Anti-EpCAM antibody [E144] (<a href='/en-us/products/primary-antibodies/epcam-antibody-e144-ab32392'>ab32392</a>) at 1/2000 dilution
Lane 1:
Wild-type A431 cell lysate at 20 µg
Lane 2:
EPCAM knockout A431 cell lysate at 20 µg
Lane 2:
Western blot - Human EPCAM knockout A-431 cell line (ab261902)
Lane 3:
MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 4:
HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Predicted band size: 35 kDa
Observed band size: 40 kDa
false
- WB
Lab
Western blot - Human EPCAM knockout A-431 cell line (AB261902)
Lanes 1 - 4 : Merged signal (red and green). Green - ab224826 observed at 35 kDa. Red - loading control ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) observed at 55kDa.
ab224826 was shown to react with EpCAM in A431 wild-type cells in Western blot. Loss of signal was observed when EpCAM knockout sample was used. A431 wild-type and EpCAM knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab224826 and ab52866 (Rabbit anti-alpha Tubulin antibody [EP1332Y]) overnight at 4° at a 1 in 400 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-EpCAM antibody [4A7] - C-terminal (<a href='/en-us/products/primary-antibodies/epcam-antibody-4a7-c-terminal-ab224826'>ab224826</a>) at 1/400 dilution
Lane 1:
Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2:
EPCAM knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 2:
Western blot - Human EPCAM knockout A-431 cell line (ab261902)
Lane 3:
MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 4:
HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Predicted band size: 35 kDa
Observed band size: 35 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human EPCAM knockout A-431 cell line (AB261902)
Knockout achieved by CRISPR/Cas9; X = 2 bp deletion; Frameshift : 99.58%
- NGS
Supplier Data
Next Generation Sequencing - Human EPCAM knockout A-431 cell line (AB261902)
2 bp deletion after Cys47 of the WT protein
Complementary Products
Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
EpCAM facilitates signaling pathways that impact cellular processes like proliferation and differentiation. It is not a part of a larger protein complex but interacts directly with other cellular machinery to transmit intracellular signals. EpCAM influences stem cell behavior and is important in tissue homeostasis assisting in both normal regenerative processes and wound healing.
Pathways
EpCAM participates in regulatory mechanisms involving the Wnt/β-catenin signaling pathway which affects gene expression and cell fate decisions. Through its action EpCAM modulates components like E-cadherin which is important for cell adhesion and the maintenance of the epithelial layer. It indirectly influences pathways relating to MYC a transcription factor that regulates cell cycle progression and apoptosis.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com