Human SMARCA4 (BRG1) knockout HEK-293T cell line (ab255432)
Overview
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Product name
Human SMARCA4 (BRG1) knockout HEK-293T cell line
See all BRG1 lysates -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 4 and 7 bp deletion in exon 4 -
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~80% -
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
Transcriptional coactivator cooperating with nuclear hormone receptors to potentiate transcriptional activation. Component of the CREST-BRG1 complex, a multiprotein complex that regulates promoter activation by orchestrating a calcium-dependent release of a repressor complex and a recruitment of an activator complex. In resting neurons, transcription of the c-FOS promoter is inhibited by BRG1-dependent recruitment of a phospho-RB1-HDAC repressor complex. Upon calcium influx, RB1 is dephosphorylated by calcineurin, which leads to release of the repressor complex. At the same time, there is increased recruitment of CREBBP to the promoter by a CREST-dependent mechanism, which leads to transcriptional activation. The CREST-BRG1 complex also binds to the NR2B promoter, and activity-dependent induction of NR2B expression involves a release of HDAC1 and recruitment of CREBBP. Belongs to the neural progenitors-specific chromatin remodeling complex (npBAF complex) and the neuron-specific chromatin remodeling complex (nBAF complex). During neural development a switch from a stem/progenitor to a post-mitotic chromatin remodeling mechanism occurs as neurons exit the cell cycle and become committed to their adult state. The transition from proliferating neural stem/progenitor cells to post-mitotic neurons requires a switch in subunit composition of the npBAF and nBAF complexes. As neural progenitors exit mitosis and differentiate into neurons, npBAF complexes which contain ACTL6A/BAF53A and PHF10/BAF45A, are exchanged for homologous alternative ACTL6B/BAF53B and DPF1/BAF45B or DPF3/BAF45C subunits in neuron-specific complexes (nBAF). The npBAF complex is essential for the self-renewal/proliferative capacity of the multipotent neural stem cells. The nBAF complex along with CREST plays a role regulating the activity of genes essential for dendrite growth. SMARCA4/BAF190A may promote neural stem cell self-renewal/proliferation by enhancing Notch-dependent proliferative signals, while concurrently making the neural stem cell insensitive to SHH-dependent differentiating cues (By similarity). Also involved in vitamin D-coupled transcription regulation via its association with the WINAC complex, a chromatin-remodeling complex recruited by vitamin D receptor (VDR), which is required for the ligand-bound VDR-mediated transrepression of the CYP27B1 gene. Acts as a corepressor of ZEB1 to regulate E-cadherin transcription and is required for induction of epithelial-mesenchymal transition (EMT) by ZEB1. -
Tissue specificity
Colocalizes with ZEB1 in E-cadherin-negative cells from established lines, and stroma of normal colon as well as in de-differentiated epithelial cells at the invasion front of colorectal carcinomas (at protein level). -
Involvement in disease
Defects in SMARCA4 are the cause of rhabdoid tumor predisposition syndrome type 2 (RTPS2) [MIM:613325]. RTPS2 is a familial cancer syndrome predisposing to renal or extrarenal malignant rhabdoid tumors and to a variety of tumors of the central nervous system, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumors. Rhabdoid tumors are the most aggressive and lethal malignancies occurring in early childhood. -
Sequence similarities
Belongs to the SNF2/RAD54 helicase family.
Contains 1 bromo domain.
Contains 1 helicase ATP-binding domain.
Contains 1 helicase C-terminal domain.
Contains 1 HSA domain. -
Post-translational
modificationsPhosphorylated upon DNA damage, probably by ATM or ATR. -
Cellular localization
Nucleus. - Information by UniProt
Associated products
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KO cell lysates
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KO cell pellets
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255432 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 185 kDa.
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| Notes |
|---|
|
WB
Use at an assay dependent concentration. Predicted molecular weight: 185 kDa. |
Images
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Western blot - Human SMARCA4 (BRG1) knockout HEK-293T cell line (ab255432)All lanes : Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/1000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SMARCA4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 185 kDa
Observed band size: 185 kDaFalse colour image of Western blot: Anti-BRG1 antibody [EPNCIR111A] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab110641 was shown to bind specifically to BRG1. A band was observed at 185 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SMARCA4 knockout cell line ab255432 (knockout cell lysate ab263853). To generate this image, wild-type and SMARCA4 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) 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.
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Western blot - Human SMARCA4 (BRG1) knockout HEK293T cell line (ab255432)All lanes : Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SMARCA4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 185 kDa
Observed band size: 185 kDaLanes 1- 2: Merged signal (red and green). Green - ab110641 observed at 185 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.
ab110641 was shown to react with SMARCA4 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255432 (knockout cell lysate ab263853) was used. Wild-type HEK-293T and SMARCA4 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. ab110641 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 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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Western blot - Human SMARCA4 (BRG1) knockout HEK293T cell line (ab255432)All lanes : Anti-BRG1 antibody [EPR3912] (ab108318) at 1/10000 dilution
Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SMARCA4 knockout HEK-293T cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 185 kDa
Observed band size: 185 kDaLanes 1- 2: Merged signal (red and green). Green - ab108318 observed at 185 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.
ab108318 was shown to react with SMARCA4 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255432 (knockout cell lysate ab263853) was used. Wild-type HEK-293T and SMARCA4 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. ab108318 and Anti-Vinculin antibody [VIN-54] overnight at 4°C at a 1 in 10000 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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Sanger Sequencing - Human SMARCA4 knockout HEK293T cell line (ab255432)Allele-1: 7 bp deletion in exon 4
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Sanger Sequencing - Human SMARCA4 knockout HEK293T cell line (ab255432)Allele-2: 1 bp deletion in exon 4.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab255432 has not yet been referenced specifically in any publications.