Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line
- Advanced Validation
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- WB
Lab
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (AB265972)
Blocking and diluting buffer and concentration : 3% NFDM/TBST
Lanes 1-3 : Merged signal (red and green). Green - ab259817 observed at 51 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab259817 Anti-TNF Receptor I antibody [EPR23742-65] was shown to specifically react with TNF Receptor I in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265972 (knockout cell lysate ab257751) was used. Wild-type and TNF Receptor I knockout samples were subjected to SDS-PAGE. ab259817 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight 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 10000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-TNF Receptor I antibody [EPR23742-65] (<a href='/en-us/products/primary-antibodies/tnf-receptor-i-antibody-epr23742-65-ab259817'>ab259817</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2:
TNFRSF1A (TNF Receptor I) knockout HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Lane 2:
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (ab265972)
Lane 3:
A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
All lanes:
Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution
Predicted band size: 50 kDa
Observed band size: 50-70 kDa
false
- WB
Lab
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (AB265972)
False colour image of Western blot : Anti-TNFRSF1A antibody staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, the antibody was shown to bind specifically to TNFRSF1A. A band was observed at 33/63 kDa in wild-type HeLa cell lysates with no signal observed at this size in TNFRSF1A knockout cell line ab265972 (knockout cell lysate ab257751). To generate this image, wild-type and TNFRSF1A 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 fluorescent western blot (TBS-based) blocking solution 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Anti-TNFRSF1A antibody at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (ab265972)
false
- WB
Lab
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (AB265972)
Lanes 1-4 : Merged signal (red and green). Green - anti-TNF-R1 Rabbit monoclonal antibody observed at 51 kDa. Red - loading control, ab8245 observed at 36 kDa.
Anti-TNF-R1 Rabbit monoclonal antibody and anti-GPADH antibody [6C5] - Loading control (ab8245) were incubated overnight at 4oC at a 1/1000 and 1/20000 dilution respectively before imaging. Blots were developed with Goat anti-Rabbig IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10,000 dilution for 1 hour at room temperature before imaging.
All lanes:
Anti-TNF-R1 Rabbit monoclonal antibody at 1/1000 dilution
Lane 1:
Human wild-type HeLa cell lysate
Lane 2:
TNFRSF1A knockout HeLa cell lysate
Lane 2:
Western blot - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (ab265972)
Lane 3:
Human brain cell lysate
Lane 4:
HL-60 cell lysate
false
- Sanger seq
Unknown
Sanger Sequencing - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (AB265972)
Allele-1 : 2 bp deletion in exon 2.
- Sanger seq
Unknown
Sanger Sequencing - Human TNFRSF1A (TNF Receptor I) knockout HeLa cell line (AB265972)
Allele-2 : 1 bp deletion in exon 2.
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Cell Lines & Lysates
AB264958
Human CASP8 (Caspase-8) knockout HeLa cell line
Advanced Validation
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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
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.
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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