Human MVP knockout HeLa cell line (ab264817)
Overview
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Product name
Human MVP knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 17 bp deletion in exon 6 and 1 bp deletion in exon 6 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: 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 -80ºC. Storage at -80ºC may result in loss of viability.
1. Thaw the vial in 37ºC water bath 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 (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37ºC 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 for confluency (80-90% confluence) within 48 hours.
- 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.
Click here to view the Mammalian cell tissue culture protocol
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~90% -
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 -
Antibiotic resistance
Puromycin 1.00µg/ml -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% DMSO, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Required for normal vault structure. Vaults are multi-subunit structures that may act as scaffolds for proteins involved in signal transduction. Vaults may also play a role in nucleo-cytoplasmic transport. Down-regulates INFG-mediated STAT1 signaling and subsequent activation of JAK. Down-regulates SRC activity and signaling through MAP kinases. -
Tissue specificity
Present in most normal tissues. Higher expression observed in epithelial cells with secretory and excretory functions, as well as in cells chronically exposed to xenobiotics, such as bronchial cells and cells lining the intestine. Overexpressed in many multidrug-resistant cancer cells. -
Sequence similarities
Contains 9 MVP (vault) repeats. -
Domain
MVP 3 mediates interaction with PTEN.
MVP 4 mediates interaction with PARP4. -
Post-translational
modificationsPhosphorylated on Tyr residues after EGF stimulation.
Dephosphorylated by PTPN11. -
Cellular localization
Cytoplasm. Nucleus > nuclear pore complex. 5% found in the nuclear pore complex. Translocates from the nucleus to the cytoplasm upon EGF treatment. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
Our Abpromise guarantee covers the use of ab264817 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. Predicted molecular weight: 99 kDa. |
Images
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Western blot - Human MVP knockout HeLa cell line (ab264817)All lanes : Anti-MVP antibody [EPR23594-106] (ab273093) at 1/1000 dilution
Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 2 : MVP knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 3 : Caco-2 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 99 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab273093 observed at 110 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab273093 Anti-MVP antibody [EPR23594-106] was shown to specifically react with MVP in wild-type HeLa cells in Western blot. Significant decrease (8.7 % of intensity compared to the WT band) of signal was observed when MVP knockout cell line ab264817 (knockout cell lysate ab257544) was used.
ab273093 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4? overnight 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 10000 dilution for 1 hour at room temperature before imaging. -
Western blot - Human MVP knockout HeLa cell line (ab264817)All lanes : Anti-MVP antibody [EPR13226(B)] (ab177145) at 1/1000 dilution
Lane 1 : Wild-type HeLa lysate
Lane 2 : MVP knockout HeLa lysate
Lane 3 : A549 lysate
Lane 4 : MOLT-4 lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 99 kDaLanes 1-4: Merged signal (red and green). Green - ab177145 observed at 99 kDa. Red - loading control ab8245 observed at 37 kDa.
ab177145 Anti-MVP antibody [EPR13226(B)] was shown to specifically react with MVP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264817 (knockout cell lysate ab257544) was used. Wild-type and MVP knockout samples were subjected to SDS-PAGE. ab177145 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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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Western blot - Human MVP knockout HeLa cell line (ab264817)All lanes : Anti-MVP antibody [EPR13227(B)] (ab175239) at 1/2000 dilution
Lane 1 : Wild-type HeLa lysate
Lane 2 : MVP knockout HeLa lysate
Lane 3 : A549 lysate
Lane 4 : MOLT-4 lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 99 kDaLanes 1-4: Merged signal (red and green). Green - ab175239 observed at 110 kDa. Red - loading control ab8245 observed at 37 kDa.
ab175239 Anti-MVP antibody [EPR13227(B)] was shown to specifically react with MVP in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264817 (knockout cell lysate ab257544) was used. Wild-type and MVP knockout samples were subjected to SDS-PAGE. ab175239 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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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Sanger Sequencing - Human MVP knockout HeLa cell line (ab264817)Allele-1: 17 bp deletion in exon 6.
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Sanger Sequencing - Human MVP knockout HeLa cell line (ab264817)Allele-2: 1 bp deletion in exon 6.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
References (0)
ab264817 has not yet been referenced specifically in any publications.
