Human RRAGD (Rag D) knockout HEK-293T cell line
- Advanced Validation
- What is this?
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RRAGD KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: 232 bp deletion in exon 1.
View Alternative Names
DKFZp761H171, FLJ44176, OTTHUMP00000016853, RRAGD_HUMAN, Rag D, Rag D protein, RagDRRAGD, Ras related GTP binding D, Ras-related GTP-binding protein D, bA11D8.2.1
- WB
Lab
Western blot - Human RRAGD (Rag D) knockout HEK-293T cell line (AB266287)
Lanes 1 - 4 : Merged signal (red and green). Green - ab187679 observed at 48 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.
ab187679 was shown to react with Rag D in wild-type HEK-293T cells in Western blot with loss of signal observed in Rragd knockout cell line ab266287 (Rragd knockout cell lysate ab259091). Wild-type HEK-293T and Rragd knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab187679 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes:
Western blot - Anti-Rag D antibody - N-terminal (<a href='/en-us/products/primary-antibodies/rag-d-antibody-n-terminal-ab187679'>ab187679</a>) at 1/2000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
RRAGD knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human RRAGD (Rag D) knockout HEK-293T cell line (ab266287)
Lane 3:
Jurkat cell lysate at 20 µg
Lane 4:
HeLa cell lysate at 20 µg
Predicted band size: 46 kDa
Observed band size: 48 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human RRAGD (Rag D) knockout HEK-293T cell line (AB266287)
Homozygous : 232 bp deletion in exon1
Reactivity data
Product details
Recommended control: Human wild-type HEK293T cell line (ab255449). 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.
We will provide viable cells that proliferate on revival.
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.
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
Rag D participates in the mTORC1 complex a protein complex that senses nutrient availability. It acts as a molecular switch signaling cells to grow or stop growth based on amino acid presence. Rag D facilitates mTORC1 activation by recruiting it to the lysosomal surface where amino acids act as a trigger. This function allows Rag D to play a significant role in cell growth division and metabolism.
Pathways
Rag D integrates into the amino acid sensing pathway directly affecting the mTOR signaling pathway important for cellular growth and homeostasis. Rag D interacts with other Rag proteins such as Rag A and Rag C to form heterodimers that regulate mTORC1 activity. This interaction ensures Rag D can sense and respond to intracellular nutrient levels contributing to homeostatic balance.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
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Cell Lines & Lysates
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Advanced Validation
- 0
- 0
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
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