ASS1 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 3 and Insertion of the selection cassette in exon 3.
Images
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 3 and Insertion of the selection cassette in exon 3
Alternative names
ASS, ASSA, ASSY_HUMAN, Argininosuccinate synthase, Argininosuccinate synthase 1, Argininosuccinate synthetase 1, CTLN1, Citrulline--aspartate ligase
Recommended products
ASS1 KO cell line available to order. KO validated by Immunocytochemistry, Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 3 and Insertion of the selection cassette in exon 3.
Key facts
- Cell type
- HeLa
- Species or organism
- Human
- Tissue
- Cervix
- Form
- Liquid
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
- Mutation description
- Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 3 and Insertion of the selection cassette in exon 3
- Disease
- Adenocarcinoma
- Concentration
Properties
- Gene name
- ASS1
- Gene editing type
- Knockout
- Gene editing method
- CRISPR technology
- Knockout validation
- Immunocytochemistry, Sanger Sequencing, Western blot
Quality control
- STR analysis
- CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
- Biosafety level
- EU: 2 US: 2
- Adherent/suspension
- Adherent
- Gender
- Female
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.
Storage
- Shipped at conditions
- Dry Ice
- Appropriate short-term storage conditions
- -196°C
- Appropriate long-term storage conditions
- -196°C
Notes
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.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
ASS1 or argininosuccinate synthase 1 is an enzyme that plays an important role in the urea cycle. It catalyzes the conversion of citrulline and aspartate into argininosuccinate using ATP in the process. The ASS1 protein is commonly expressed in the liver where it actively participates in the detoxification of ammonia. It has a molecular mass of approximately 46 kDa. Some alternate names for this protein include ASS and ASS-1.
- Biological function summary
Argininosuccinate synthase 1 contributes significantly to the synthesis of arginine an essential amino acid through its conversion activities. The enzyme operates as a homotrimer a complex consisting of three identical subunits. This enzymatic activity is indispensable in maintaining the balance of nitrogen and ammonia within the body particularly important for liver and kidney functions.
- Pathways
ASS1 is a critical component of both the urea cycle and the nitric oxide metabolism pathway. The urea cycle helps in detoxifying ammonia by converting it into urea which is excreted in urine. In nitric oxide metabolism ASS1 links with nitric oxide synthase to facilitate the use of arginine in producing nitric oxide a vital signaling molecule. The pathway engagement partners include proteins like citrin which supplies aspartate for the reaction.
- Associated diseases and disorders
ASS1 has implications in citrullinemia and hepatocellular carcinoma. Citrullinemia results from mutations in the ASS1 gene leading to defective urea cycle function and ammonia accumulation. In cancer particularly hepatocellular carcinoma low expression of ASS1 correlates with poor prognosis as cancer cells often exhibit arginine auxotrophy. Connections through these conditions involve interactions with enzymes such as arginase which also influences arginine availability.
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6 product images
Western blot - Human ASS1 knockout HeLa cell line (ab264989)
Lanes 1- 2: Merged signal (red and green). Green - Anti-ASS1 antibody [EPR12399(B)] - C-terminal ab170900 observed at 47 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.Anti-ASS1 antibody [EPR12399(B)] - C-terminal ab170900 was shown to react with ASS1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264989 (knockout cell lysate Human ASS1 knockout HeLa cell lysate ab257143) was used. Wild-type HeLa and ASS1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-ASS1 antibody [EPR12399(B)] - C-terminal ab170900 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (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-ASS1 antibody [EPR12399(B)] - C-terminal (Anti-ASS1 antibody [EPR12399(B)] - C-terminal ab170900) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: ASS1 knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human ASS1 knockout HeLa cell line (ab264989)
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDa
Immunocytochemistry/ Immunofluorescence - Human ASS1 knockout HeLa cell line (ab264989)
Anti-ASS1 antibody [EPR12398] ab170952 staining ASS1 in wild-type HeLa cells (top panel) and ASS1 knockout HeLa cells (ab264989) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-ASS1 antibody [EPR12398] ab170952 at 1/100 dilution and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Immunocytochemistry/ Immunofluorescence - Human ASS1 knockout HeLa cell line (ab264989)
Anti-ASS1 antibody [2B10] ab124465 staining ASS1 in wild-type HeLa cells (top panel) and ASS1 knockout HeLa cells (ab264989) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with Anti-ASS1 antibody [2B10] ab124465 at 1/1000 dilution and Anti-beta Tubulin antibody - Loading Control ab6046 (Rabbit polyclonal to beta Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117) at 2 μg/ml (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
Cell Culture - Human ASS1 knockout HeLa cell line (ab264989)
Representative images of ASS1 knockout HeLa cells, low and high confluency examples (top left and right respectively) and wild-type HeLa cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS XL Core microscope.
Sanger Sequencing - Human ASS1 knockout HeLa cell line (ab264989)
Allele-1: 1 bp insertion in exon 3.
Sanger Sequencing - Human ASS1 knockout HeLa cell line (ab264989)
Allele-2: Insertion of the selection cassette in exon 3.
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