CAVIN1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 191 bp insertion in exon 1 and 5 bp deletion in exon 1.
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Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Sanger Sequencing
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 191 bp insertion in exon 1 and 5 bp deletion in exon 1
Alternative names
2310075E07Rik, AW546441, CGL4, Cavin, Cavin-1, FKSG13, FLJ90031, MGC118550, OTTMUSP00000002049, PTRF_HUMAN, Polymerase I and transcript release factor, RNA polymerase I and transcript release factor, RP23-279L23.6, TTF I interacting peptide 12
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CAVIN1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 191 bp insertion in exon 1 and 5 bp deletion in exon 1.
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Sanger Sequencing
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 191 bp insertion in exon 1 and 5 bp deletion in exon 1
- Antibiotic resistance
- Puromycin 1µg/mL
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- CAVIN1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing
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.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
PTRF also known as Cavin-1 is a protein with a mass of approximately 43 kDa. It functions primarily as a regulator of caveolae formation and stability. Caveolae are small flask-shaped invaginations in the plasma membrane. PTRF interacts directly with caveolin proteins to facilitate caveolae assembly. It is largely expressed in adipose tissue muscle tissue and endothelial cells indicating a significant role in cellular processes in these cells.
- Biological function summary
PTRF facilitates various processes like signal transduction and lipid metabolism due to its role in caveolae function. As part of the caveolae complex it is essential for maintaining structural integrity and organization. PTRF interacts with other proteins such as cavin family members and caveolins aiding in cellular response to mechanochemical stimuli. By regulating the caveolae PTRF influences activities including endocytosis and potocytosis.
- Pathways
PTRF is critical in cholesterol homeostasis and endothelial nitric oxide synthase (eNOS) signaling pathways. It associates with caveolin-1 modulating these pathways to balance lipid composition and promote endothelial function. The interaction between PTRF and caveolin-1 enhances nitric oxide production which is important for vascular tone regulation and cellular stress response. This positions PTRF as a regulator in cardiovascular and metabolic pathways.
- Associated diseases and disorders
Defects or deficiencies in PTRF can lead to disorders such as lipodystrophy and muscular dystrophy. The absence or malfunction of PTRF disrupts normal caveolae structure leading to impaired lipid storage and muscle function. PTRF's interaction with caveolin-1 connects it to cardiovascular diseases as caveolin-1 mutations contribute to the pathogenesis of cardiac dysfunction. Understanding PTRF's role offers insights into therapeutic strategies for these conditions.
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6 product images
Western blot - Human PTRF knockout HeLa cell line (ab265435)
False colour image of Western blot: Anti-PTRF antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to PTRF. A band was observed at 55 kDa in wild-type HeLa cell lysates with no signal observed at this size in PTRF knockout cell line ab265435 (knockout cell lysate Human PTRF knockout HeLa cell lysate ab257620). To generate this image, wild-type and PTRF 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 °C. 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: Anti-PTRF antibody at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: PTRF knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human PTRF knockout HeLa cell line (ab265435)
Lane 3: A431 cell lysate at 20 µg
Lane 4: HL-60 cell lysate at 20 µg
Performed under reducing conditions.
Sandwich ELISA - Human PTRF knockout HeLa cell line (ab265435)
Sandwich ELISA of Human PTRF Antibody Pair - BSA and Azide free ab312881 with the capture antibody dilution at 2 µg/mL and detector antibody dilution at 0.5 µg/mL.
Interpolated concentrations of native PTRF in human control wild type HeLa cell and PTRF knockout HeLa cell based on 25 µg/mL extract loads. The concentrations of PTRF were measured in duplicate and interpolated from the PTRF standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean PTRF concentration was determined to be 281.5 pg/mL in wild type HeLa extract (Human wild-type HeLa cell line ab255928) and undetectable in PTRF knockout HeLa extract (Human PTRF knockout HeLa cell line ab265435).
Sanger Sequencing - Human PTRF knockout HeLa cell line (ab265435)
Allele-1: 5 bp deletion in exon 1.
Sanger Sequencing - Human PTRF knockout HeLa cell line (ab265435)
Allele-2: 191 bp insertion in exon 1.
Immunocytochemistry/ Immunofluorescence - Human PTRF knockout HeLa cell line (ab265435)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PTRF KO HeLa (PTRF knockout human cervical adenocarcinoma epithelial cel), ab265435 cells labelling PTRF with Alexa Fluor® 647 Anti-PTRF antibody [EPR27006-67] ab318173 at 1/50 (10.0 ug/ml) dilution (Green).
Confocal image showing membranous and cytoplasmic staining in wildtype HeLa cells and showing no staining in PTRF knockout HeLa cells (shown in magenta). The counterstain was observed in green. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).Alexa Fluor® 488 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195887 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 488) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
Immunocytochemistry/ Immunofluorescence - Human PTRF knockout HeLa cell line (ab265435)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PTRF KO HeLa (PTRF knockout human cervical adenocarcinoma epithelial cells), ab265435 cells labelling PTRF with Alexa Fluor® 488 Anti-PTRF antibody [EPR27006-67] ab320701 at 1/50 (10.0 ug/ml) dilution (Green).
Confocal image showing membranous and cytoplasmic staining in wildtype HeLa cells and showing no staining in PTRF knockout HeLa cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).
Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).
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