Human NF1 (Neurofibromin) knockout HeLa cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human NF1 (Neurofibromin) knockout HeLa cell line (AB264725)
Lanes 1-3 : Merged signal (red and green). Green - ab238142 observed at 319 kDa. Red - loading control ab7291 observed at 50 kDa.
ab238142 Recombinant Anti-NF1 antibody [EPR22989-68] was shown to specifically react with NFIB in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264725 (knockout cell lysate ab258533) was used. Wild-type and NF1 knockout samples were subjected to SDS-PAGE. ab238142 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C 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 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-Neurofibromin antibody [EPR22989-68] (<a href='/en-us/products/primary-antibodies/neurofibromin-antibody-epr22989-68-ab238142'>ab238142</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
NFIB knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human NF1 (Neurofibromin) knockout HeLa cell line (ab264725)
Lane 3:
HAP1 cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution
Predicted band size: 319 kDa
Observed band size: 319 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human NF1 (Neurofibromin) knockout HeLa cell line (AB264725)
Homozygous : 1 bp deletion in exon 1.
Complementary Products
Primary Antibodies
AB238142
Anti-Neurofibromin antibody [EPR22989-68]
RabMAb|Recombinant|KO Validated
![Western blot - Anti-Neurofibromin antibody [EPR22989-68] (AB238142)](https://content.abcam.com/products/images/neurofibromin-antibody-epr22989-68-ab238142--western-blot-img6363.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 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
Neurofibromin regulates pathways involved in cell growth and development. It does not form part of a large multiprotein complex but interacts with microtubules and other signaling proteins to carry out its regulatory functions. Neurofibromin primarily affects pathways that manage cell cycle progression and it plays a critical role in inhibiting uncontrolled cell growth ensuring proper cellular homeostasis.
Pathways
Neurofibromin fits into the Ras-MAPK pathway where it controls the activity of Ras an important regulatory protein involved in signal transduction related to cell division and differentiation. Neurofibromin's action on Ras directly impacts the activation of the MAPK pathway which is important in mediating cellular responses to growth signals. This protein also interacts functionally with proteins like SOS1 another regulator of Ras emphasizing its role in fine-tuning signal cascades that ensure balanced cellular function.
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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