Human CAV1 (Caveolin-1) knockout HeLa cell line
- Advanced Validation
- What is this?
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(0 Publication)
- ICC/IF
Unknown
Immunocytochemistry/ Immunofluorescence - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
ab17052 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (ab255371) (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 ab17052 at 1/500 dilution 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
Unknown
Immunocytochemistry/ Immunofluorescence - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
ab192869 staining Caveolin-1 in wild-type HeLa cells (top panel) and CAV1 knockout HeLa cells (ab255371) (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 ab192869 at 1/500 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).
- WB
Lab
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Lanes 1 - 4 : Merged signal (red and green). Green - ab32577 observed at 20 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab32577 was shown to react with Caveolin-1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab255371 (knockout cell lysate ab263806) was used. Wild-type and Caveolin-1 knockout samples were subjected to SDS-PAGE. ab32577 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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-Caveolin-1 antibody [E249] - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-e249-caveolae-marker-ab32577'>ab32577</a>) at 1/1000 dilution
Lane 1:
A431 cell lysate at 20 µg
Lane 2:
A549 cell lysate at 20 µg
Lane 2:
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)
Lane 3:
Wild-type HeLa cell lysate at 20 µg
Lane 4:
CAV1 knockout HeLa cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution
Predicted band size: 20 kDa
Observed band size: 20 kDa,37 kDa
false
- WB
Lab
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Lanes 1 - 4 : Merged signal (red and green). Green - ab193893 observed at 20 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab193893 was shown to react with Caveolin-1 in wild-type HeLa cells in Western blot with loss of signal observed in CAV1 knockout cell line ab255371 (CAV1 knockout cell lysate ab263806). Wild-type HeLa and CAV1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab193893 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature. Blots were developed with Optiblot ECL reagent (ab133456) before imaging.
All lanes:
Western blot - HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (<a href='/en-us/products/primary-antibodies/hrp-caveolin-1-antibody-e249-caveolae-marker-ab193893'>ab193893</a>) at 1/5000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
CAV1 knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)
Lane 3:
A431 cell lysate at 20 µg
Lane 4:
A549 cell lysate at 20 µg
Predicted band size: 20 kDa
Observed band size: 20 kDa
false
Exposure time: 90s
- WB
Unknown
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Lanes 1 - 4 : Merged signal (red and green). Green - ab192869 observed at 20 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab192869 was shown to react with Caveolin-1 in wild-type HeLa. Loss of signal was observed when knockout cell line ab255371 (knockout cell lysate ab263806) was used. Wild-type and Caveolin-1 knockout samples were subjected to SDS-PAGE. ab192869 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 10000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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-Caveolin-1 antibody [EPR15554] - N-terminal - Caveolae Marker (<a href='/en-us/products/primary-antibodies/caveolin-1-antibody-epr15554-n-terminal-caveolae-marker-ab192869'>ab192869</a>) at 1/10000 dilution
Lane 1:
A431 cell lysate at 20 µg
Lane 2:
A549 cell lysate at 20 µg
Lane 2:
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)
Lane 3:
Wild-type HeLa cell lysate at 20 µg
Lane 4:
CAV1 knockout HeLa cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution
Predicted band size: 20 kDa
Observed band size: 37 kDa
false
- Flow Cyt
Lab
Flow Cytometry - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Flow cytometry overlay histogram showing wild-type HeLa (green line) and CAV1 knockout HeLa cells (ab255371) stained with ab192869 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab192869) (1x106 in 100μl at 0.04 μg/ml) for 30 min at 22°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 22°C.
Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HeLa - black line CAV1 knockout HeLa - 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.
- Sanger seq
Unknown
Sanger Sequencing - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Allele-2 : Insertion of the selection cassette in exon 1.
- Sanger seq
Unknown
Sanger Sequencing - Human CAV1 (Caveolin-1) knockout HeLa cell line (AB255371)
Allele-1 : 1 bp insertion in exon 1.
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, 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.
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
Caveolae provide a platform for various signaling pathways and involve caveolin-1 as a major component. Caveolin-1 interacts with multiple signaling molecules such as G-protein coupled receptors and Src family kinases to modulate signal transduction. This protein forms part of a larger caveolar complex contributing to cellular processes including endocytosis and lipid regulation. Its presence as a marker in caveolae highlights its significance in cellular functions.
Pathways
Caveolin-1 influences the insulin signaling and nitric oxide (NO) signaling pathways. In the insulin signaling pathway caveolin-1 interacts with insulin receptors to modulate glucose uptake. It also associates with eNOS (endothelial nitric oxide synthase) in the NO signaling pathway impacting vascular function. These relationships highlight its role in cellular communication and regulatory mechanisms within the human body.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
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'.
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