Human DNMT3A knockout HeLa cell line
- Advanced Validation
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DNMT3A KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon 4 and 1 bp insertion in exon 4.
View Alternative Names
DNA (cytosine 5) methyltransferase 3 alpha, DNA (cytosine-5)-methyltransferase 3A, DNA MTase HsaIIIA, DNA cytosine methyltransferase 3A2, DNA methyltransferase 3 alpha, DNA methyltransferase 3a, DNA methyltransferase HsaIIIA, DNM3A_HUMAN, DNMT, DNMT 3a, DNMT3A2, M.HsaIIIA, MCMT, OTTHUMP00000201149, TBRS
- WB
Lab
Western blot - Human DNMT3A knockout HeLa cell line (AB261793)
Lanes 1-4 : Merged signal (red and green). Green - ab227823 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.
ab227823 Anti-Dnmt3a antibody [EPR18455-94] was shown to specifically react with Dnmt3a in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261793 (knockout cell lysate ab257128) was used. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab227823 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4° 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 20000 dilution for 1 hour at room temperature before imaging.
All lanes:
Western blot - Anti-Dnmt3a antibody [EPR18455-94] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr18455-94-ab227823'>ab227823</a>) at 1/1000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
Dnmt3a knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human DNMT3A knockout HeLa cell line (ab261793)
Lane 3:
HEK-293 cell lysate at 20 µg
Lane 4:
Daudi cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution
Predicted band size: 102 kDa
Observed band size: 125 kDa
false
- WB
Supplier Data
Western blot - Human DNMT3A knockout HeLa cell line (AB261793)
Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS. The samples were run on a Bis-Tris gel.
False colour image of Western blot : Anti-Dnmt3a antibody ab307503 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, ab307503 was shown to bind specifically to Dnmt3a. A band was observed at 125 kDa in wild-type Hela cell lysates whereas no signal observed at this size in Dnmt3a knockout cell line (ab261793). To generate this image, wild-type and Dnmt3a knockout Hela cell lysates were analyzed. First, samples were run on a SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in 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) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-Dnmt3a antibody [EPR26805-273] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr26805-273-ab307503'>ab307503</a>) at 1/1000 dilution
Lane 1:
Wild-type Hela whole cell lysate 20 μg
Lane 2:
Western blot - Human DNMT3A knockout HeLa cell line (ab261793)
Lane 2:
Dnmt3a knockout Hela whole cell lysate 20 μg
Lane 3:
Mouse brain tissue lysate 20 μg
Secondary
Lanes 1 - 3:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Lanes 1 - 3:
Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution
Observed band size: 125 kDa
false
- WB
Lab
Western blot - Human DNMT3A knockout HeLa cell line (AB261793)
Lanes 1-4 : Merged signal (red and green). Green - ab188470 observed at 125 kDa. Red - loading control ab8245 observed at 37 kDa.
ab188470 Anti-Dnmt3a antibody [EPR18455] was shown to specifically react with Dnmt3a in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab261793 (knockout cell lysate ab257128) was used. Wild-type and Dnmt3a knockout samples were subjected to SDS-PAGE. ab188470 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 2000 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.
All lanes:
Western blot - Anti-Dnmt3a antibody [EPR18455] (<a href='/en-us/products/primary-antibodies/dnmt3a-antibody-epr18455-ab188470'>ab188470</a>) at 1/2000 dilution
Lane 1:
Wild-type HeLa cell lysate at 20 µg
Lane 2:
Dnmt3a knockout HeLa cell lysate at 20 µg
Lane 2:
Western blot - Human DNMT3A knockout HeLa cell line (ab261793)
Lane 3:
HEK-293 cell lysate at 20 µg
Lane 4:
Daudi cell lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution
Predicted band size: 102 kDa
Observed band size: 125 kDa
false
- Sanger seq
Unknown
Sanger Sequencing - Human DNMT3A knockout HeLa cell line (AB261793)
Allele-2 : 1 bp insertion in exon 4.
- Sanger seq
Unknown
Sanger Sequencing - Human DNMT3A knockout HeLa cell line (AB261793)
Allele-1 : 1 bp deletion in exon 4.
Reactivity data
Product details
Recommended control: Human wild-type HeLa cell line (ab255928). 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.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
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
Complementary Products
Primary Antibodies
AB188470
Anti-Dnmt3a antibody [EPR18455]
RabMAb|Recombinant|KO Validated|Advanced Validation
![Immunocytochemistry/ Immunofluorescence - Anti-Dnmt3a antibody [EPR18455] (AB188470)](https://content.abcam.com/products/images/dnmt3a-antibody-epr18455-ab188470--immunocytochemistry-immunofluorescence-img103616.jpg)
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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