Human PUS1 knockout HEK-293T cell line (ab266091)
Overview
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Product name
Human PUS1 knockout HEK-293T cell line -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 41 bp deletion in exon 2 and 47 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
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.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
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. Based on cell count, seed cells in an appropriate cell culture 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.
4. Incubate the culture at 37°C incubator with 5% CO2. 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.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Kidney -
Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Converts specific uridines to PSI in a number of tRNA substrates. Acts on positions 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. Involved in regulation of nuclear receptor activity possibly through pseudouridylation of SRA1 RNA. -
Tissue specificity
Widely expressed. High levels of expression found in brain and skeletal muscle. -
Involvement in disease
Defects in PUS1 are a cause of myopathy with lactic acidosis and sideroblastic anemia type 1 (MLASA1) [MIM:600462]; also known as mitochondrial myopathy and sideroblastic anemia. MLASA is a rare autosomal recessive oxidative phosphorylation disorder specific to skeletal muscle and bone marrow. -
Sequence similarities
Belongs to the tRNA pseudouridine synthase TruA family. -
Cellular localization
Mitochondrion and Nucleus. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab266091 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 47 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 47 kDa. |
Images
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All lanes : Anti-PUS1 antibody [EPR20181] (ab203010) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PUS1 knockout HEK-293T cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Daudi cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab203010 observed at 45 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab203010 was shown to react with PUS1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266091 (knockout cell lysate ab258158) was used. Wild-type HEK-293T and PUS1 knockout HEK-293T 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. ab203010 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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 (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.
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Allele-1: 47 bp deletion in exon2
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Allele-2: 41 bp deletion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266091 has not yet been referenced specifically in any publications.