Human DNMT3A knockout HeLa cell lysate (ab257128)
Overview
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Product name
Human DNMT3A knockout HeLa cell lysate -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon4 and 1 bp insertion in exon4. -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.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.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab260160 - Human DNMT3A knockout HeLa cell lysate 1 x 100µg ab255929 - Human wild-type HeLa cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
Target
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Function
Required for genome wide de novo methylation and is essential for the establishment of DNA methylation patterns during development. DNA methylation is coordinated with methylation of histones. It modifies DNA in a non-processive manner and also methylates non-CpG sites. May preferentially methylate DNA linker between 2 nucleosomal cores and is inhibited by histone H1. Plays a role in paternal and maternal imprinting. Required for methylation of most imprinted loci in germ cells. Acts as a transcriptional corepressor for ZNF238. Can actively repress transcription through the recruitment of HDAC activity. -
Tissue specificity
Highly expressed in fetal tissues, skeletal muscle, heart, peripheral blood mononuclear cells, kidney, and at lower levels in placenta, brain, liver, colon, spleen, small intestine and lung. -
Sequence similarities
Belongs to the C5-methyltransferase family.
Contains 1 ADD domain.
Contains 1 GATA-type zinc finger.
Contains 1 PHD-type zinc finger.
Contains 1 PWWP domain. -
Domain
The PWWP domain is essential for targeting to pericentric heterochromatin. -
Post-translational
modificationsSumoylated; sumoylation disrupts the ability to interact with histone deacetylases (HDAC1 and HDAC2) and repress transcription. -
Cellular localization
Nucleus. Cytoplasm. Accumulates in the major satellite repeats at pericentric heterochromatin. - Information by UniProt
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Alternative names
- DNA (cytosine 5) methyltransferase 3 alpha
- DNA (cytosine 5) methyltransferase 3A
- DNA (cytosine-5)-methyltransferase 3A
see all
Associated products
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KO cell lines
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab257128 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: 102 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 102 kDa. |
Images
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Western blot - Human DNMT3A knockout HeLa cell lysate (ab257128)
Lane 1: Wild-type HeLa cell lysate (20 µg)
Lane 2: DNMT3A knockout HeLa cell lysate (20 µg)
Lane 3: HEK-293 cell lysate (20 µg)
Lane 4: Daudi cell lysate (20 µg)
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°C 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.
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Western blot - Human DNMT3A knockout HeLa cell lysate (ab257128)Lane 1: Wild-type HeLa cell lysate (20 µg)
Lane 2: DNMT3A knockout HeLa cell lysate (20 µg)
Lane 3: HEK-293 cell lysate (20 µg)
Lane 4: Daudi cell lysate (20 µg)
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.
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Sanger Sequencing - Human DNMT3A knockout HeLa cell lysate (ab257128)Allele-1: 1 bp deletion in exon4
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Sanger Sequencing - Human DNMT3A knockout HeLa cell lysate (ab257128)Allele-2: 1 bp insertion in exon4
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab257128 has not yet been referenced specifically in any publications.