Human BRD3 knockout HEK-293T cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human BRD3 knockout HEK-293T cell line (AB266793)
Lanes 1-4 : Merged signal (red and green). Green - ab50818 observed at 100 kDa. Red - loading control ab181602 observed at 36 kDa.
ab50818 Anti-BRD3 antibody [2088C3a] was shown to specifically react with BRD3 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266793 (knockout cell lysate ab258335) was used. Wild-type and BRD3 knockout samples were subjected to SDS-PAGE. ab50818 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4° at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-BRD3 antibody [2088C3a] (<a href='/en-us/products/primary-antibodies/brd3-antibody-2088c3a-ab50818'>ab50818</a>) at 1/500 dilution
Lane 1:
Wild-type HEK293T cell lysate at 20 µg
Lane 2:
BRD3 knockout HEK293T cell lysate at 20 µg
Lane 2:
Western blot - Human BRD3 knockout HEK-293T cell line (ab266793)
Lane 3:
Jurkat cell lysate at 20 µg
Lane 4:
HeLa cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution
Predicted band size: 80 kDa
Observed band size: 100 kDa
false
- WB
Lab
Western blot - Human BRD3 knockout HEK-293T cell line (AB266793)
Blocking and dilution buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
The identity of the lower MW band at approximately 50kDa is unknown.
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
False colour image of Western blot : Anti-BRD3 antibody [EPR23743-226] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 loading control staining at 1/20000 dilution, shown in red.
In Western blot, ab300106 was shown to bind specifically to BRD3. A band was observed at 100 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in BRD3 knockout cell line ab266793 (knockout cell lysate ab258335).
To generate this image, wild-type and BRD3 knockout HEK-293 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 1/10000 dilution.
All lanes:
Western blot - Anti-BRD3 antibody [EPR23743-226] (<a href='/en-us/products/primary-antibodies/brd3-antibody-epr23743-226-ab300106'>ab300106</a>) at 1/1000 dilution
Lane 1:
Wild type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg
Lane 2:
BRD3 knockout HEK-293T whole cell lysate at 20 µg
Lane 3:
HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg
Secondary
Lanes 1 - 3:
Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed at 1/10000 dilution
Lanes 1 - 3:
Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed at 1/10000 dilution
Predicted band size: 80 kDa
Observed band size: 100 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human BRD3 knockout HEK-293T cell line (AB266793)
Homozygous : 1 bp deletion in exon 4
- Cell Culture
Unknown
Cell Culture - Human BRD3 knockout HEK-293T cell line (AB266793)
Representative images of BRD3 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using an EVOS M5000 microscope.
- ICC/IF
Lab
Immunocytochemistry/ Immunofluorescence - Human BRD3 knockout HEK-293T cell line (AB266793)
Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized wildtype HEK-293T(human embryonic kidney epithelial cell); BRD3 knockout HEK-293T, ab266793 cells labelling BRD3 with ab325539 at 1/1000 (0.512 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).
Confocal image showing nuclear staining in wildtype HEK-293T cells (shown in green), showing no staining in BRD3 knockout HEK-293T cells. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).
Complementary Products
Primary Antibodies
AB300107
Anti-BRD3 antibody [EPR23743-226] (BSA and Azide free)
RabMAb|Recombinant|KO Validated
![Western blot - Anti-BRD3 antibody [EPR23743-226] (BSA and Azide free) (AB300107)](https://content.abcam.com/products/images/brd3-antibody-epr23743-226-bsa-and-azide-free-ab300107--western-blot-img449783.jpg)
- 0
- 0
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
Zygosity
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.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
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