Human CARM1 knockout HEK-293T cell line (ab266557)
Overview
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Product name
Human CARM1 knockout HEK-293T cell line
See all CARM1 lysates -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette 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, 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.
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
Methylates (mono- and asymmetric dimethylation) the guanidino nitrogens of arginyl residues in several proteins involved in DNA packaging, transcription regulation, pre-mRNA splicing, and mRNA stability. Recruited to promoters upon gene activation together with histone acetyltransferases from EP300/P300 and p160 families, methylates histone H3 at 'Arg-17' (H3R17me), forming mainly asymmetric dimethylarginine (H3R17me2a), leading to activate transcription via chromatin remodeling. During nuclear hormone receptor activation and TCF7L2/TCF4 activation, acts synergically with EP300/P300 and either one of the p160 histone acetyltransferases NCOA1/SRC1, NCOA2/GRIP1 and NCOA3/ACTR or CTNNB1/beta-catenin to activate transcription. During myogenic transcriptional activation, acts together with NCOA3/ACTR as a coactivator for MEF2C. During monocyte inflammatory stimulation, acts together with EP300/P300 as a coactivator for NF-kappa-B. Acts as coactivator for PPARG, promotes adipocyte differentiation and the accumulation of brown fat tissue. Plays a role in the regulation of pre-mRNA alternative splicing by methylation of splicing factors. Also seems to be involved in p53/TP53 transcriptional activation. Methylates EP300/P300, both at 'Arg-2142', which may loosen its interaction with NCOA2/GRIP1, and at 'Arg-580' and 'Arg-604' in the KIX domain, which impairs its interaction with CREB and inhibits CREB-dependent transcriptional activation. Also methylates arginine residues in RNA-binding proteins PABPC1, ELAVL1 and ELAV4, which may affect their mRNA-stabilizing properties and the half-life of their target mRNAs. -
Tissue specificity
Overexpressed in prostate adenocarcinomas and high-grade prostatic intraepithelial neoplasia. -
Sequence similarities
Belongs to the protein arginine N-methyltransferase family. -
Post-translational
modificationsAuto-methylated on Arg-550. Methylation enhances transcription coactivator activity. Methylation is required for its role in the regulation of pre-mRNA alternative splicing.
Phosphorylation at Ser-216 interferes with S-adenosyl-L-methionine binding and strongly reduces methyltransferase activity (By similarity). Phosphorylation at Ser-216 is strongly increased during mitosis, and decreases rapidly to a very low, basal level after entry into the G1 phase of the cell cycle. Phosphorylation at Ser-216 may promote location in the cytosol. -
Cellular localization
Nucleus. Cytoplasm. Mainly nuclear during the G1, S and G2 phases of the cell cycle. Cytoplasmic during mitosis, after breakup of the nuclear membrane. - 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 ab266557 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: 65 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 65 kDa. |
Images
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All lanes : Anti-CARM1 antibody [EPR23678-113] (ab243638) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 2 : CARM1 knockout HEK-293T (human embryonic kidney epithelial cell), whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) at 1/10000 dilution
Predicted band size: 65 kDa
Observed band size: 65 kDaBlocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
Lanes 1 - 4: Merged signal (red and green). Green - ab243638 observed at 65 kDa. Red - loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) observed at 36 kDa.
ab243638 was shown to react with CARM1 in wild-type HEK-293T cells in western blot with loss of signal observed in CARM1 knockout cell line ab266557 (CARM1 knockout cell lysate ab257871). Wild-type and CARM1 knockout HEK-293T cell lysates were subjected to SDS-PAGE.
ab243638 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.
Boiled samples are used in this blot.
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All lanes : Anti-CARM1 antibody (ab128851) at 1/500 dilution
Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : CARM1 knockout HEK293T cell lysate
Lane 3 : HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 65 kDa
Observed band size: 66 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab128851 observed at 66 kDa. Red - loading control ab8245 observed at 36 kDa.
ab128851 Anti-CARM1 antibody was shown to specifically react with CARM1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266557 (knockout cell lysate ab257871) was used. Wild-type and CARM1 knockout samples were subjected to SDS-PAGE. ab128851 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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.
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Homozygous: Insertion of the selection cassette in exon2
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab266557 has not yet been referenced specifically in any publications.