Human RHOT1 (MIRO1) knockout HeLa cell line
- Advanced Validation
- What is this?
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- WB
Lab
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] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, 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 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 (ab216772) and Goat anti-Rabbit IgG H&L (IRDye®680RD) preabsorbed (ab216777) at 1/20000 dilution.
All lanes:
Western blot - Anti-MIRO1 antibody [CL1083] (<a href='/en-us/products/primary-antibodies/miro1-antibody-cl1083-ab188029'>ab188029</a>) 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
Predicted band size: 71 kDa
Observed band size: 71 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human RHOT1 (MIRO1) knockout HeLa cell line (AB265792)
Homozygous : 1 bp insertion in exon 1.
- WB
Supplier Data
Western blot - Human RHOT1 (MIRO1) knockout HeLa cell line (AB265792)
Blocking and diluting buffer and concentration : 5% NFDM/TBST.
In Western blot, 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 (ab181602) staining at 1/200000 dilution.
All lanes:
Western blot - Anti-MIRO1 antibody [MJF-D29136-4] (<a href='/en-us/products/primary-antibodies/miro1-antibody-mjf-d29136-4-ab319154'>ab319154</a>) 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
Secondary
All lanes:
Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution
Observed band size: 80 kDa,36 kDa
false
Exposure time: 48s
Complementary Products
Primary Antibodies
AB320019
Anti-MIRO1 antibody [MJF-D29136-145] - BSA and Azide free (Detector)
Recombinant|RabMAb|KO Validated
![Sandwich ELISA - Anti-MIRO1 antibody [MJF-D29136-145] - BSA and Azide free (Detector) (AB320019)](https://content.abcam.com/products/images/miro1-antibody-mjf-d29136-145-bsa-and-azide-free-detector-ab320019--sandwich-elisa-img433963.jpg)
- 0
- 0
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
Zygosity
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
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.
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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