RHOT1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 1.
Images
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, Homozygous: 1 bp insertion in exon 1
Alternative names
ARHT 1, FLJ11040, FLJ12633, MIRO1_HUMAN, Mitochondrial Rho 1, Mitochondrial Rho GTPase 1, Rac-GTP-binding protein-like protein, Ras homolog family member T1, Ras homolog gene family member T1, Rhot 1, hMiro-1, mitochondrial Rho (MIRO) GTPase 1
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RHOT1 KO cell line available to order. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion 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, Homozygous: 1 bp insertion in exon 1
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- RHOT1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Sanger Sequencing
- Zygosity
- Homozygous
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
MIRO1 also known as RhoT1 is a mitochondrial Rho GTPase with a molecular mass of approximately 69 kDa. It functions as a pivotal component in mitochondrial transport and dynamics. This protein localizes to the outer mitochondrial membrane and is highly expressed in various tissues particularly in the brain and muscles. MIRO1 facilitates the anchoring of mitochondria to microtubule motors enabling their movement within the cell which is essential for maintaining proper energy distribution.
- Biological function summary
MIRO1 plays a significant role in regulating mitochondrial trafficking along the cytoskeleton. It forms part of a protein complex with TRAK1/2 kinesin and dynein motor proteins. MIRO1's presence in this complex allows it to modulate mitochondrial distribution and dynamics which are essential for neural development and function. It also influences mitochondrial shape and interaction with the endoplasmic reticulum impacting intracellular calcium homeostasis.
- Pathways
MIRO1 is an integral component of pathways involving mitochondrial transport and cellular energy homeostasis. It actively participates in the regulation of calcium signaling pathways alongside related proteins like TRAK1. Additionally MIRO1's involvement in the maintenance of mitochondrial dynamics links it to mitophagy pathways. These pathways regulate the removal of damaged mitochondria a vital process for cellular health and longevity.
- Associated diseases and disorders
MIRO1 has connections to neurodegenerative conditions such as Parkinson's disease and Alzheimer's disease. These conditions correlate with impaired mitochondrial trafficking and dynamics. MIRO1 interacts with proteins such as PINK1 and Parkin which contribute to mitochondrial quality control and are involved in the pathogenesis of these diseases. The disruption of MIRO1 function can lead to mitochondrial dysfunction and energy deficits which are underlying factors in these neurodegenerative processes.
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3 product images
Western blot - Human RHOT1 (MIRO1) knockout HeLa cell line (ab265792)
False colour image of Western blot: Anti-MIRO1 antibody [CL1083] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-MIRO1 antibody [CL1083] ab188029 was shown to bind specifically to MIRO1. A band was observed at 71 kDa in wild-type HeLa cell lysates with no signal observed at this size in RHOT1 knockout cell line ab265792 (knockout cell lysate Human RHOT1 (MIRO1) knockout HeLa cell lysate ab258635). To generate this image, wild-type and RHOT1 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-Mouse IgG H&L (IRDye®800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye®680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
All lanes: Western blot - Anti-MIRO1 antibody [CL1083] (Anti-MIRO1 antibody [CL1083] ab188029) at 1/500 dilution
Lane 1: Wild-type HeLa cell lysate at 40 µg
Lane 2: RHOT1 knockout HeLa cell lysate at 40 µg
Lane 2: Western blot - Human RHOT1 (MIRO1) knockout HeLa cell line (ab265792)
Lane 3: HEK-293 cell lysate at 20 µg
Lane 4: A431 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 71 kDa
Observed band size: 71 kDa
Sanger Sequencing - Human RHOT1 (MIRO1) knockout HeLa cell line (ab265792)
Homozygous: 1 bp insertion in exon 1.
Western blot - Human RHOT1 (MIRO1) knockout HeLa cell line (ab265792)
Blocking and diluting buffer and concentration: 5% NFDM/TBST.
p>In Western blot, Anti-MIRO1 antibody [MJF-D29136-4] ab319154 was shown to bind specifically to RHOT1. Target of interest was observed at 80 kDa band in wild-type Hela cell lysates (lane 1) with no signal observed at this size in RHOT1 knockout cell line (lane 2) (lane 2, knockout cell line ab265792).In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.
All lanes: Western blot - Anti-MIRO1 antibody [MJF-D29136-4] (Anti-MIRO1 antibody [MJF-D29136-4] ab319154) at 1/1000 dilution
Lane 1: HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2: Western blot - Human RHOT1 (MIRO1) knockout HeLa cell line (ab265792) at 20 µg
SecondaryAll lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Performed under reducing conditions.
Observed band size: 80 kDa, 36 kDa
Exposure time: 48s
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