Human PRKRA (PACT) knockout HEK-293T cell line
- Advanced Validation
- What is this?
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(0 Publication)
- WB
Unknown
Western blot - Human PRKRA (PACT) knockout HEK-293T cell line (AB266806)
Lanes 1-4 : Merged signal (red and green). Green - ab31967 observed at 36 kDa. Red - loading control ab8245 observed at 36 kDa.
ab31967 Anti-PACT (PKR activating protein) / PRKRA antibody was shown to specifically react with PACT in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266806 (knockout cell lysate ab258141) was used. Wild-type and PACT knockout samples were subjected to SDS-PAGE. ab31967 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours 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-PACT (PKR activating protein) / PRKRA antibody (<a href='/en-us/products/primary-antibodies/pact-pkr-activating-protein-prkra-antibody-ab31967'>ab31967</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
PRKRA knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human PRKRA (PACT) knockout HEK-293T cell line (ab266806)
Lane 3:
K-562 cell lysate at 20 µg
Lane 4:
HepG2 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 1/10000 dilution
Predicted band size: 34 kDa
Observed band size: 36 kDa
false
- WB
Lab
Western blot - Human PRKRA (PACT) knockout HEK-293T cell line (AB266806)
Lanes 1-4 : Merged signal (red and green). Green - ab75749 observed at 36 kDa. Red - loading control ab8245 observed at 36 kDa.
ab75749 Anti-PACT (PKR activating protein) / PRKRA antibody [EPR3224] was shown to specifically react with PACT in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266806 (knockout cell lysate ab258141) was used. Wild-type and PACT knockout samples were subjected to SDS-PAGE. ab75749 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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-PACT (PKR activating protein) / PRKRA antibody [EPR3224] (<a href='/en-us/products/primary-antibodies/pact-pkr-activating-protein-prkra-antibody-epr3224-ab75749'>ab75749</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T cell lysate at 20 µg
Lane 2:
PRKRA knockout HEK-293T cell lysate at 20 µg
Lane 2:
Western blot - Human PRKRA (PACT) knockout HEK-293T cell line (ab266806)
Lane 3:
K-562 cell lysate at 20 µg
Lane 4:
HepG2 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 1/10000 dilution
Predicted band size: 34 kDa
Observed band size: 36 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human PRKRA (PACT) knockout HEK-293T cell line (AB266806)
Allele-2 : 1 bp deletion in exon 1.
- Sanger seq
Unknown
Sanger Sequencing - Human PRKRA (PACT) knockout HEK-293T cell line (AB266806)
Allele-1 : 2 bp deletion in exon1
Complementary Products
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.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
PACT involves in the cellular stress response signaling. It serves as an activator for PKR a kinase that becomes active during viral infections and stress. PACT forms complexes with PKR facilitating its activation and subsequent initiation of downstream signaling cascades. It contributes significantly to the regulation of protein synthesis and cell growth by modulating these pathways. In conditions of stress PACT ensures adequate PKR activation for host defense.
Pathways
PACT is key in the interferon signaling pathway and the stress response pathway. It partners with PKR to suppress protein synthesis through phosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) which slows down protein production during cellular stress. This interaction places PACT at a junction of antiviral defense and stress adaptation with PKR serving as an essential protein involved in these pathways.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com