Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)
Overview
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Product name
Human CAV1 (Caveolin-1) knockout HeLa cell line
See all Caveolin-1 lysates -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 1 and Insertion of the selection cassette in exon 1 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: Flow Cyt, ICC, WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255448). 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 (High Glucose) + 10% 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 2x104 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 2x104 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.
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8, 12 CSF1PO: 9, 10 -
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
May act as a scaffolding protein within caveolar membranes. Interacts directly with G-protein alpha subunits and can functionally regulate their activity (By similarity). Involved in the costimulatory signal essential for T-cell receptor (TCR)-mediated T-cell activation. Its binding to DPP4 induces T-cell proliferation and NF-kappa-B activation in a T-cell receptor/CD3-dependent manner. Recruits CTNNB1 to caveolar membranes and may regulate CTNNB1-mediated signaling through the Wnt pathway. -
Tissue specificity
Expressed in muscle and lung, less so in liver, brain and kidney. -
Involvement in disease
Defects in CAV1 are the cause of congenital generalized lipodystrophy type 3 (CGL3) [MIM:612526]; also called Berardinelli-Seip congenital lipodystrophy type 3 (BSCL3). Congenital generalized lipodystrophies are autosomal recessive disorders characterized by a near absence of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early onset of diabetes. -
Sequence similarities
Belongs to the caveolin family. -
Post-translational
modificationsThe initiator methionine for isoform Beta is removed during or just after translation. The new N-terminal amino acid is then N-acetylated. -
Cellular localization
Golgi apparatus membrane. Cell membrane. Membrane > caveola. Membrane raft. Colocalized with DPP4 in membrane rafts. Potential hairpin-like structure in the membrane. Membrane protein of caveolae. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
- Anti-Caveolin-1 antibody [7C8] (ab17052)
- Anti-Caveolin-1 antibody [EPR15554] - N-terminal (ab192869)
- HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab193893)
- Anti-Caveolin-1 antibody [E249] - BSA and Azide free (ab230262)
- Anti-Caveolin-1 antibody [EPR15554] - BSA and Azide free (ab240332)
- Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577)
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255371 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 |
|---|---|---|
| Flow Cyt |
Use at an assay dependent concentration.
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| ICC |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration. Predicted molecular weight: 20 kDa.
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| Notes |
|---|
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Flow Cyt
Use at an assay dependent concentration. |
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ICC
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 20 kDa. |
Images
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Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)All lanes : HRP Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab193893) at 1/5000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CAV1 knockout HeLa cell lysate
Lane 3 : A431 cell lysate
Lane 4 : A549 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 20 kDa
Observed band size: 20 kDa
Exposure time: 90 secondsLanes 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.
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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). -
Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)All lanes : Anti-Caveolin-1 antibody [EPR15554] - N-terminal (ab192869) at 1/10000 dilution
Lane 1 : A431 cell lysate
Lane 2 : A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : CAV1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 20 kDa
Additional bands at: 37 kDa (possible Loading Control)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.
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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.
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Western blot - Human CAV1 (Caveolin-1) knockout HeLa cell line (ab255371)All lanes : Anti-Caveolin-1 antibody [E249] - Caveolae Marker (ab32577) at 1/1000 dilution
Lane 1 : A431 cell lysate
Lane 2 : A549 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : CAV1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 20 kDa
Additional bands at: 37 kDa (possible Loading Control)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.
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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). -
Sanger Sequencing - Human CAV1 knockout HeLa cell line (ab255371)Allele-1: 1 bp insertion in exon 1.
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Sanger Sequencing - Human CAV1 knockout HeLa cell line (ab255371)Allele-2: Insertion of the selection cassette in exon 1.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab255371 has not yet been referenced specifically in any publications.